Methods for modulation of immune responses using humanized immunoglobulins reactive with B7-1 molecules

ABSTRACT

The invention relates to humanized anti-B7-2 and anti-B7-1 antibodies, wherein each comprise a variable region of non-human origin and at least a portion of an immunoglobulin of human origin. The invention also pertains to methods of treatment for various autoimmune diseases, transplant rejection, inflammatory disorders and infectious diseases by administering humanized anti-B7-2 and/or anti-B7-1 antibodies.

RELATED APPLICATION(S)

This application is a Continuation-In-Part of application Ser. No.09/249,011, now U.S. Pat. No. 6,972,125, filed Feb. 12, 1999, and is adivisional of application Ser. No. 09/339,596, now U.S. Pat. No.6,913,747, filed Jun. 24, 1999, the entire teaching of both of which arehereby incorporated by reference.

BACKGROUND OF THE INVENTION

Antigen specific T-cell activation and the initiation of an immuneresponse depend initially on the interaction of the T-cell receptor(TCR) complex with the peptide/major histocompatibility complex (MHC)present on antigen presenting cells (APC). B7 molecules, B7-1 and B7-2,are molecules which are present on APCs. A “co-stimulatory” signal,provided by the interaction of B7-1 and B7-2 on the APC with theirligands CD28 and CTLA4 on T-cells, is required to complete T-cellactivation and the subsequent regulation of an immune response. A needexists to regulate the B7-1 and B7-2 pathway, referred to as theB7:CD28/CTLA4 pathway. A further need exists to develop treatments fordiseases that are affected by this pathway.

SUMMARY OF THE INVENTION

The invention relates to humanized immunoglobulins having bindingspecificity for B7 molecules. In particular, the invention includes ahumanized immunoglobulin having binding specificity for B7-2 or B7-1,wherein the immunoglobulin comprises an antigen binding region ofnon-human origin (e.g. rodent) and at least a portion of human origin(e.g. a human constant region such as an IgG constant region and/or ahuman framework region). In one embodiment, the human constant region ofeither the humanized anti-B7-2 or humanized anti-B7-1 immunoglobulin canalso contain a mutation that reduces the effector function of thehumanized immunoglobulin. The humanized B7-2 immunoglobulin describedherein can compete with murine 3D1 for binding to B7-2. Similarly, thehumanized B7-1 immunoglobulin described herein can compete with murine1F1 for binding to B7-1. In particular embodiments, the antigen bindingregion of the humanized B7-2 immunoglobulin is derived from the 3D1monoclonal antibody, and the antigen binding region of the humanized137-1 immunoglobulin is derived from the 1F1 monoclonal antibody.

The humanized immunoglobulins of the present invention can comprise aconstant region of human origin and an antigen binding region, whereinthe antigen binding region of non-human origin comprises one or morecomplementarity determining regions (CDRs) of rodent origin (e.g.,derived from 3D1 monoclonal antibody) that binds to B7-2, or one or moreCDRs of rodent origin (e.g., derived from 1F1 monoclonal antibody) thatbinds to B7-1, and the portion of an immunoglobulin of human origin isderived from a human framework region (FR).

The antigen binding region of the humanized B7-2 antibody can furthercomprise a light chain and a heavy chain, wherein the light and heavychain each have three CDRs derived from the 3D1 antibody. The FR of thelight chain for the humanized B7-2 antibody can be derived, for example,from the light chain of the human H₂F antibody and the heavy chain canbe derived, for example, from the heavy chain of the human III-2Rantibody. In a particular embodiment, the invention is a humanizedimmunoglobulin having binding specificity for B7-2 that is derived fromthe cell line deposited with the American Type Culture Collection(A.T.C.C.) Accession No. CRL-12524.

The antigen binding region of the humanized B7-1 antibody can comprise alight chain and a heavy chain, wherein the light and heavy chain eachhave three CDRs derived from the 1F1 antibody. The FR of the humanizedB7-1 light and heavy chain can be derived, for example, from the lightand heavy chains of the human III-2R antibody. In a particularembodiment, the invention is a humanized immunoglobulin having bindingspecificity for B7-1 that is derived from the cell line deposited withthe American Type Culture Collection (A.T.C.C.) Accession No.PTA-263.

The invention also embodies a humanized immunoglobulin having a bindingspecificity for B7-1 or B7-2 comprising a heavy chain and/or a lightchain. In one embodiment, the humanized immunoglobulin has bindingspecificity for B7-2 and the light chain comprises at least one CDR(e.g., CDR1, CDR2 and CDR3) derived from an antibody of non-human originwhich binds B7-2 and a FR derived from a light chain of human origin(e.g., H2F antibody); and the heavy chain comprises at least one CDR(e.g., CDR1, CDR2 and CDR3) derived from an antibody of non-human originwhich binds B7-2 and a FR region derived from a heavy chain of humanorigin (e.g., the human III-2R antibody). In another embodiment, thehumanized immunoglobulin has binding specificity for the B7-1 and thelight and/or heavy chain comprise at least one CDR (e.g., CDR1, CDR2 andCDR3) derived from an antibody of non-human origin which binds B7-1 anda FR derived from a light and/or heavy chain of human origin which bindsB7-1 and a FR derived from a light and/or heavy chain of human origin(e.g., III-2R). The immunoglobulins can further comprise CDR1, CDR2 andCDR3 for the light or heavy chain having the amino acid sequence setforth herein or an amino acid substantially the same as the amino acidsequence such that the antibody specifically binds to B7-2 or B7-1. Thepresent invention also relates to humanized immunoglobulin light chainsand to humanized immunoglobulin heavy chains. The invention furtherrelates to isolated nucleic acids comprising a sequence which encodesthe humanized immunoglobulins of the present invention (e.g., a singlechain antibody), and the invention also relates to isolated nucleicacids that comprise a sequence which encodes the B7-2 or B7-1 humanizedimmunoglobulin light chain or heavy chain.

One embodiment of the invention is a humanized immunoglobulin lightchain having binding specificity for B7-2 comprising CDR1, CDR2 and/orCDR3 of the light chain of murine 3D1 antibody, and a human light chainFR (e.g., H₂F antibody). Similarly, the invention also comprises ahumanized B7-1 immunoglobulin light chain having binding specificity forB7-1 comprising CDR1, CDR2 and/or CDR3 of the light chain of the murine1F1 antibody, and a human light chain FR (e.g., III-2R antibody).Another embodiment is a humanized B7-2 or B7-1 immunoglobulin lightchain that comprises a variable region shown in FIG. 2B (SEQ ID NO: 8)or the variable region shown in FIG. 7B (SEQ ID NO: 28). The inventionalso relates to an isolated nucleic acid sequence that encodes ahumanized variable light chain specific for B7-2 or B7-1 that comprisesa nucleic acid sequence shown in FIG. 2B (SEQ ID NO: 7) or FIG. 7B (SEQID NO.: 27), respectively, a nucleic acid sequence that encodes theamino acid sequence shown in FIG. 2B (SEQ ID NO: 8) or FIG. 7B (SEQ IDNO: 28), respectively, a nucleic acid sequence which hybridizes theretounder stringent hybridization conditions, or a nucleic acid sequencewhich is the complement thereof.

Another embodiment of the invention is a humanized immunoglobulin heavychain that is specific for B7-2 and comprises CDR1, CDR2 and/or CDR3 ofthe heavy chain of the 3D1 antibody, and a human heavy chain FR (e.g.,I2R antibody). Similarly, the invention also relates to a B7-1 humanizedimmunoglobulin heavy chain that is specific for B7-1 and comprises CDR1,CDR2, and/or CDR3 of the heavy chain of the 1F1 antibody, and a humanheavy chain FR (e.g., III-2R antibody). The invention pertains to ahumanized immunoglobulin heavy chain that comprises a variable regionshown in FIG. 2A (SEQ ID NO: 6) or FIG. 7A (SEQ ID NO: 26). Theinvention also pertains to an isolated nucleic acid sequence thatencodes a humanized variable heavy chain specific for B7-2 thatcomprises a nucleic acid sequence shown in FIG. 2A (SEQ ID NO: 5), anisolated nucleic acid sequence that encodes the amino acid sequenceshown in FIG. 2A (SEQ ID NO: 6), a nucleic acid sequence whichhybridizes thereto under stringent hybridization conditions, or anucleic acid sequence which is the complement thereof. The inventionrelates to an isolated nucleic acid sequence that encodes a humanizedvariable heavy chain specific for B7-1 that comprises a nucleic acidsequence shown in FIG. 7A (SEQ ID NO: 25), an isolated nucleic acidsequence that encodes the amino acid sequence shown in FIG. 7A (SEQ IDNO: 26), a nucleic acid sequence which hybridizes thereto understringent hybridization conditions, or a nucleic acid sequence which isthe complement thereof.

In particular, an embodiment of the invention is a humanizedimmunoglobulin which specifically binds to B7-2 and comprises ahumanized light chain comprising three light chain CDRs from the mouse3D1 antibody, and a light chain variable region framework sequence froma human immunoglobulin light chain, and a humanized heavy chaincomprising three heavy chain CDRs from the mouse 3D1 antibody, and aheavy chain variable region framework sequence from a humanimmunoglobulin heavy chain. The mouse 3D1 antibody can further have amature light chain variable domain, such as the mature light chainvariable domain shown in FIG. 1B (SEQ ID NO.: 4) and a mature heavychain variable domain such as the mature heavy chain variable regionshown in FIG. 1A (SEQ ID NO.: 2).

Another embodiment of the invention is a humanized immunoglobulin whichspecifically binds to B7-1 and comprises a humanized light chaincomprising three light chain CDRs from the mouse 1F1 antibody, and alight chain variable region framework sequence from a humanimmunoglobulin light chain, and a humanized heavy chain comprising threeheavy chain CDRs from the mouse 1F1 antibody, and a heavy chain variableregion framework sequence from a human immunoglobulin heavy chain.Similarly, the mouse 1F1 antibody can further have a mature light chainvariable domain, such as the mature light chain variable domain shown inFIG. 6B (SEQ ID NO.: 24) and a mature heavy chain variable domain suchas the mature heavy chain variable region shown in FIG. 6A (SEQ ID NO.:22).

The invention includes an expression vector that comprises a fused genewhich encodes humanized B7-1 and/or B7-2 immunoglobulin light and/orheavy chains. The gene comprises a nucleotide sequence encoding a CDRderived from a light and/or heavy chain of a non-human antibody havingbinding specificity for B7-2 and/or B7-1 (e.g., murine 3D1 or 1F1antibody, respectively) and a FR derived from a light and/or heavy chainof human origin.

The present invention also relates to a host cell comprising a nucleicacid of the present invention, including one or more constructscomprising nucleic acid of the present invention. In one embodiment, theinvention encompasses a host cell comprising a first B7-2 recombinantnucleic acid that encodes a humanized B7-2 immunoglobulin light chainand a second B7-2 recombinant nucleic acid that encodes a humanized B7-2immunoglobulin heavy chain. The first B7-2 nucleic acid comprises anucleotide sequence encoding at least one CDR derived from the lightchain of murine 3D1 antibody and a FR derived from a light chain ofhuman origin. The second B7-2 nucleic acid comprises a nucleotidesequence encoding at least one CDR derived from the heavy chain ofmurine 3D1 antibody and a FR derived from a heavy chain of human origin.In another embodiment, the invention encompasses a host cell comprisinga first B7-1 recombinant nucleic acid that encodes a humanized B7-1immunoglobulin light chain and a second B7-1 recombinant nucleic acidthat encodes a humanized B7-1 immunoglobulin heavy chain. The first B7-1nucleic acid comprises a nucleotide sequence encoding at least one CDRderived from the light chain of murine 1F1 antibody and a FR derivedfrom a light chain of human origin. The second B7-1 nucleic acidcomprises a nucleotide sequence encoding at least one CDR derived fromthe heavy chain of murine 1F1 antibody and a FR derived from a heavychain of human origin. The invention further relates to a host cellcomprising a vector or a nucleic acid that encodes the humanized B7-1and/or B7-2 immunoglobulins, as described herein.

The invention further pertains to methods of preparing humanizedimmunoglobulins that comprise maintaining a host cell that encodes ahumanized immunoglobulin that is specific for B7-2 or B7-1, as describedherein, under conditions appropriate for expression of a humanizedimmunoglobulin, wherein a humanized immunoglobulin chain (one or more)are expressed and a humanized immunoglobulin is produced. The methodfurther comprises the step of isolating the humanized B7-1 or B7-2immunoglobulin.

The invention encompasses methods of inhibiting the interaction of afirst cell bearing a B7-2 receptor with a second cell bearing B7-2comprising contacting the second cell with an effective amount of ahumanized B7-2 immunoglobulin, as described herein. The invention alsoencompasses methods of inhibiting the interaction of a first cellbearing a B7-1 receptor with a second cell bearing B7-1 comprisingcontacting the second cell with an effective amount of a humanized B7-1immunoglobulin, as described herein. Accordingly, the invention relatesto inhibiting both a B7-1 and B7-2 receptor with B7-1 and B7-2 ligands,comprising contacting the cells having the B7-1 and B7-2 receptors withan amount of humanized anti-B7-1 and B7-2 immunoglobulins. Thus, theinvention pertains to various methods of treatment. The inventionincludes a method for modulating an immune response of an individual, ortreating an individual having a transplanted organ, tissue, cell or thelike comprising administering an effective amount of a humanized B7-1and/or B7-2 immunoglobulin, as described herein, with or without acarrier (e.g., pharmaceutical carrier), wherein the immune response ismodulated. The invention pertains to treating acute and/or chronictransplant rejection, for example, for a prolonged periods of time(e.g., days, months, or years). The invention also pertain to methods oftreating a disease associated with modulation of the B7-2 and/or B7-1molecules (e.g., autoimmune diseases, infectious diseases, inflammatorydisorders, systemic lupus erythematosus, diabetes mellitus, asthma,insulitis, arthritis, inflammatory bowel disease, inflammatorydermatitis, and multiple sclerosis), comprising administering to anindividual an effective amount (e.g., a therapeutically effectiveamount) of the B7-2 and/or B7-1 humanized immunoglobulins, as describedherein, with or without a carrier. Accordingly, the inventionencompasses a pharmaceutical composition comprising the B7-1 and/or B7-2humanized immunoglobulins, as described herein.

The invention also embodies methods of making a humanized immunoglobulinspecific to B7-2 from a murine antibody specific to B7-2, and/or methodsof making a humanized immunoglobulin specific to B7-1 from a murineantibody specific to B7-1. The methods comprise determining orascertaining the CDRs of an antibody of non-human origin (e.g., murineorigin) which has binding specificity for B7-2 or B7-1; obtaining ahuman antibody having a framework region amino acid sequence suitablefor grafting of the CDRs, and grafting the CDRs of an antibody ofnon-human origin into the FR of the human antibody.

The invention also relates to methods for determining the presence orabsence of B7-2 and/or B7-1 in a sample. The methods comprise obtainingthe sample to be tested, contacting the sample with the humanizedantibody specific to B7-2 and/or B7-1, or a fragment thereof,sufficiently to allow formation of a complex between B7-2 and/or B7-1and the anti-B7-2 and/or anti-B7-1 antibody, respectively, and detectingthe presence or absence of the complex formation. The presence of thecomplex indicates the presence of B7-2 and/or B7-1 in the sample.

The invention relates to methods for treating an individual having adisease comprising administering an amount (e.g., therapeuticallyeffective amount) of a humanized immunoglobulin specific to B7-1 and/oran amount (e.g., therapeutically effective amount) of a humanizedimmunoglobulin specific to B7-2. The diseases, as described herein,include, for example, autoimmune diseases, infectious diseases, asthma,inflammatory disorders, systemic lupus erythematosus, diabetes mellitus,insulitis, arthritis, inflammatory bowel disease, inflammatorydermatitis, and multiple sclerosis. This method also pertains tomodulating the immune response of an individual having a transplantedorgan, tissue, cell or the like comprising administering an effectiveamount of a humanized immunoglobulin that binds to B7-1 and/or ahumanized immunoglobulin that binds to B7-2.

The invention also pertains to methods for transplanting cells (e.g.,bone marrow, blood cells, blood components and other cells) to anindividual in need thereof comprising obtaining cells (e.g., bonemarrow, or blood cells or components) from a donor, and contacting thecells with an immunoglobulin specific to B7-1 and/or an immunoglobulinspecific to B7-2, and recipient cells, thereby obtaining a mixture. Theimmunoglobulins and the recipient cells are maintained for a period oftime sufficient for tolerance induction. The mixture (referred to as abone marrow composition or blood cell composition) is then introducedinto the individual. The recipient cells can be a lymphocyte antigen(e.g. lymphocytes that express class 1 antigens (MHCI) or peripheralblood lymphocyte (PBL)). Instead of using recipient cells, the methodalso comprise utilizing tissue, organs or cells that express MHC Class Iantigens, B7-1 and/or B7-2 molecules. The cells can be engineered toexpress recipient molecules. The cells from the donor can be bone marrowcells, or cells/components from blood (e.g., stem cells or immaturecells). The B7 immunoglobulins are in contact with the donor bone marrowand the recipient cells for a period of time that is long enough toinduce tolerance induction (e.g., about 1 to 48 hours, and, preferablyabout 36 hours). An individual in need of such a transplant is one whohas a disease that is benefitted by or treatable with a bone marrowtransplant. Such diseases, for example, are proliferative diseases (e.g.leukemia, lymphoma and cancer), anemia (e.g. sickle-cell anemia,thalassemia, and aplastic anemia) and myeloid dysplasia syndrome (MDS).

In particular, the invention includes methods for transplanting bonemarrow to an individual having a disease (e.g., proliferative diseasessuch as leukemia, lymphoma, cancer; anemia such as sickle-cell anemia,thalassemia, and aplastic anemia; and myeloid dysplasia syndrome) thatis treated with a bone marrow transplant comprising obtaining bonemarrow from a donor, and contacting the bone marrow with immunoglobulinsspecific to B7-1 and/or an immunoglobulin specific to B7-2, andrecipient cells (e.g., lymphocyte). The bone marrow, immunoglobulin(s)and recipient cells are in contact for a period of time sufficient fortolerance induction (e.g., about 1-48 hours, preferably about 36 hours).The method then comprises re-introducing the treated bone marrow to theindividual.

Advantages of the invention include the ability to regulate or modulatethe B7 co-stimulatory pathway. Manipulation of this co-stimulatorypathway with humanized anti-B7-2 and/or anti-B7-1 antibodies providemethods of treatments for various diseases. The humanized anti-B7-2 andanti-B7-1 antibodies maintain about the same specificity for respectiveB7 molecule as the corresponding murine antibody, but with a reducedimmunogenicity in humans and an extended half-life, as compared with themurine counterpart. Accordingly, the invention can advantageously beused to treat immune-related diseases/disorders, or diseases in whichthe B7-2 and/or B7-1 molecules play an important role. Particularly, theinvention relates to methods for treating autoimmune diseases, andmethods for modulating the immune response for individuals withtransplanted organs, tissue or cells.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing and other embodiments, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying figures.

FIG. 1A is a sequence listing of the heavy chain variable region nucleicacid and amino acid sequences (SEQ ID NOS: 1 and 2, respectively) of themurine 3D1 antibody, wherein the amino acid sequences of the CDRs (CDR1,CDR2 and CDR3) are underlined, and the first amino acid of the mature,heavy chain is double underlined.

FIG. 1B is a sequence listing of the light chain variable region nucleicacid and amino acid sequences (SEQ ID NOS: 3 and 4, respectively) of themurine 3D1 antibody wherein, the nucleic and amino acid sequences of theCDRs (CDR1, CDR2 and CDR3) are underlined, and the first amino acid ofthe mature light chain is double underlined.

FIG. 2A is a sequence listing of the heavy chain variable region nucleicacid and amino acid sequences (SEQ ID NOs: 5 and 6, respectively) of thehumanized 3D1 antibody, wherein the nucleic and amino acid sequences ofthe CDRs (CDR1, CDR2 and CDR3), and underlined and the first amino acidof the mature heavy chain is double underlined.

FIG. 2B contains the light chain variable region nucleic acid and aminoacid sequences (SEQ ID NOs: 7 and 8, respectively) of the humanized 3D1antibody, wherein the nucleic and amino acid sequences of CDR1, CDR2 andCDR3. The CDRs are underlined, and the first amino acid of the maturelight chain is double underlined.

FIG. 3 is a graph depicting the results of a competitive binding assay.The graph depicts the results of a competitive binding assay of murineor humanized anti-human B7-2 mAbs to CHO expressing rhB7-2 (CHO/hB7-2)on their surface. Increasing concentrations of unlabeled competitorantibodies were incubated with CHO/hB7-2 cells in the presence ofradiolabeled tracer murine anti-human B7-2 mAb and the ratio ofbound/free antibody was determined.

FIG. 4 is a graph depicting the results of a direct binding assay ofmurine or humanized anti-human B7-2 mAbs to CHO/hB7-2 cells. Increasingconcentrations of radiolabeled antibodies were incubated with CHO orCHO/hB7-2 cells and the amount of specific antibody bound to theCHO/hB7-2 cells was determined.

FIG. 5 is a graph depicting the results of a T cell proliferation assay.Increasing concentrations of murine or humanized anti-human B7-2 mAbswere added to CD28+human T cells stimulated with PMA and CHO/hB7-2 cellsand the inhibition of T cell proliferation by these mAbs was determined.

FIG. 6A is a sequence listing of the heavy chain variable region nucleicacid and amino acid sequences (SEQ ID NOS: 21 and 22, respectively) ofthe murine 1F1 antibody, wherein the amino acid sequences of the CDRs(CDR1, CDR2 and CDR3) are underlined, and the first amino acid of themature heavy chain is double underlined.

FIG. 6B is a sequence listing of the light chain variable region nucleicacid and amino acid sequences (SEQ ID NOS: 23 and 24, respectively) ofthe murine 1F1 antibody wherein, the nucleic and amino acid sequences ofthe CDRs (CDR1, CDR2 and CDR3) are underlined, and the first amino acidof the mature light chain is double underlined.

FIG. 7A is a sequence listing of the heavy chain variable region nucleicacid and amino acid sequences (SEQ ID NOs: 25 and 26, respectively) ofthe humanized 1F1 antibody (hu1F1), wherein the nucleic and amino acidsequences of the CDRs (CDR1, CDR2 and CDR3) are underlined, and thefirst amino acid of the mature heavy chain is double underlined.

FIG. 7B contains the light chain variable region nucleic acid and aminoacid sequences (SEQ ID NOs: 27 and 28, respectively) of the humanized1F1 (hu1F1) antibody, wherein the nucleic and amino acid sequences ofCDR1, CDR2 and CDR3. The CDRs are underlined, and the first amino acidof the mature light chain is double underlined.

FIG. 8 is a graph depicting the results of a competitive binding assay.The graph depicts the results of a competitive binding assay of murineor humanized anti-human B7-1 mAbs to CHO transfected with rhB7-1(CHO/hB7-1). Increasing concentrations of unlabeled competitorantibodies were incubated with CHO/hB7-1 cells in the presence ofradiolabeled tracer humanized 1F1, and the ratio of bound/free antibodywas determined.

FIG. 9A is a graph showing the Scatchard analysis of the binding ofmouse 1F1 antibodies to CHO cells transfected with rhB7-1. Radiolabeledmouse 1F1 antibodies were incubated with CHO cells transfected withrhB7-1, and the ratio of bound/free radioactivity was determined.

FIG. 9B is a graph showing the Scatchard analysis of the binding ofhumanized 1F1 antibodies to CHO cells transfected with rhB7-1.Radiolabeled humanized 1F1 antibodies were incubated with CHO cellstransfected with rhB7-1, and the ratio of bound/free radioactivity wasdetermined.

FIG. 10 is a graph depicting the results of a competitive binding assayof murine or humanized anti-human B7-1 mAbs to CHO expressing rhB7-1(CHO/hB7-1) on their surface. Increasing concentrations of unlabeledcompetitor antibodies were incubated with CHO/hB7-1 cells in thepresence of radiolabeled tracer murine anti-human B7-1 mAb and the ratioof bound/free antibody was determined.

FIG. 11 is a graph depicting the results of a direct binding assay ofmurine or humanized anti-human B7-1 mAbs to CHO/hB7-1 cells. Increasingconcentrations of radiolabeled antibodies were incubated with CHO orCHO/hB7-1 cells and the amount of specific antibody bound to theCHO/hB7-1 cells was determined.

FIG. 12 is a graph depicting the results of a T cell proliferationassay. Increasing concentrations of murine or humanized anti-human B7-1mAbs were added to CD28⁺ human T cells stimulated with PMA and CHO/hB7-1cells and the inhibition of T cell proliferation by these mAbs wasdetermined.

FIG. 13 is a graph depicting the results of a one way mixed lymphocytereaction (MLR) assay. Fixed concentrations of murine or humanizedanti-human B7-2 (IgG2.M3 isotype) or human CTLA4Ig were added to amixture of human responder and stimulator PBLs and the proliferation ofthe responder PBLs was determined on days 3, 4, and 5 by the addition ofradiolabeled thymidine.

FIG. 14 is a graph depicting the results of a one way secondary MLRassay using PBLs from a primary MLR as responders and PBLs from the sameor a different individual as in the primary MLR as stimulators. Thehumanized anti-human B7-1 mAb was added to the primary MLR only.Proliferation of the responder PBLs in the secondary MLR was determinedon days 3, 4, and 5 by the addition of radiolabeled thymidine.

FIG. 15 is a graph depicting the results of a one way secondary MLRassay using PBLs from a primary MLR as responders and PBLs from the sameor a different individual as in the primary MLR as stimulators. Thehumanized anti-human B7-2 mAb (IgG2.M3 isotype) was added to the primaryMLR only. Proliferation of the responder PBLs in the secondary MLR wasdetermined on days 3, 4, and 5 by the addition of radiolabeledthymidine.

FIG. 16 is a graph depicting the results of a one way secondary MLRassay using PBLs from a primary MLR as responders and PBLs from the sameor a different individual as in the primary MLR as stimulators. Thehumanized anti-human B7-1 and B7-2 mAbs (IgG2.M3 isotype) were added tothe primary MLR only. Proliferation of the responder PBLs in thesecondary MLR was determined on days 3, 4, and 5 by the addition ofradiolabeled thymidine.

FIG. 17 is a graph depicting the anti-tetanus response in non-humanprimates immunized with tetanus toxoid. Cynomolgus monkeys wereimmunized with purified tetanus toxoid and treated with humanizedanti-B7-1 and humanized anti-B7-2 (IgG2.M3 isotype) antibodies. Serumanti-tetanus antibody titers (IgM & IgG) were measured weekly over a 12week period.

FIG. 18 is a graph showing the serum concentration of anti-B7-1 andanti-B7-2 (IgG2.M3 isotype) mAbs at various times after administrationof an I.V. dose of 10 mg/kg.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to humanized immunoglobulins having bindingspecificity for B 7-2 or B7-1, comprising an antigen binding region ofnon-human origin and at least a portion of an immunoglobulin of humanorigin. Preferably, the humanized immunoglobulins can bind B7-2 or B7-1with an affinity of at least about 10⁷M⁻¹, preferably at least about 10⁸M⁻¹, and more preferably at least about 10⁹1M⁻¹. In one embodiment, thehumanized immunoglobulins include an antigen binding region of non-humanorigin which binds B7-2 or B7-1 and a constant region derived from ahuman constant region. The human constant region can have non-humanresidues in the framework region (FR). In another embodiment, thehumanized immunoglobulins which binds B7-2 or B7-1 comprise acomplementarity determining region (one or more) of non-human origin anda variable framework region (one or more) of human origin, andoptionally, a constant region of human origin. Optionally, the FR regionof the immunoglobulins can comprise residues of non-human origin. Forexample, the humanized immunoglobulins can comprise a heavy chain and alight chain, wherein the light chain comprises a complementaritydetermining region derived from an antibody of non-human origin whichbinds B7-2 and a framework region derived from a light chain of humanorigin, and the heavy chain comprises a complementarity determiningregion derived from an antibody of non-human origin which binds B7-2 anda framework region derived from a heavy chain of human origin. Inanother example, the humanized immunoglobulins can comprise a heavychain and a light chain, wherein the light chain comprises acomplementarity determining region derived from an antibody of non-humanorigin which binds B7-1 and a framework region derived from a lightchain of human origin, and the heavy chain comprises a complementaritydetermining region derived from an antibody of non-human origin whichbinds B7-1 and a framework region derived from a heavy chain of humanorigin. Also, the invention, individually or in a functionalcombination, embodies the light chain, the heavy chain, the variableregion, the variable light chain and the variable heavy chain.

The invention relates to a humanized B7-2 antibody that hassubstantially the same binding specificity as the murine B7-2 antibodyfrom which the humanized antibody is made, but with reducedimmunogenicity in primates (e.g., humans). Similarly, the invention alsorelates to a humanized B7-1 antibody that has substantially the samebinding specificity as the murine B7-1 antibody, respectively, fromwhich the humanized antibody is made, but with reduced immunogenicity inprimates (e.g., humans). The humanized B7-2 or B7-1 antibody can haveabout a lesser, substantially the same, or greater binding affinity asthe murine counterpart. See FIGS. 3, 4, 8, 9A and 9B.

Naturally occurring immunoglobulins have a common core structure inwhich two identical light chains (about 24 kD) and two identical heavychains (about 55 or 70 kD) form a tetramer. The amino-terminal portionof each chain is known as the variable (V) region, also referred to asthe “antigen binding” region, and can be distinguished from the moreconserved constant (C) regions of the remainder of each chain. Withinthe variable region of the light chain is a C-terminal portion known asthe J region. Within the variable region of the heavy chain, there is aD region in addition to the J region. Most of the amino acid sequencevariation in immunoglobulins is confined to three separate locations inthe V regions known as hypervariable regions or complementaritydetermining regions (CDRs) which are directly involved in antigenbinding. The variable region is the portion of the antibody that bindsto the antigen. The constant region allows for various functions such asthe ability to bind to Fc receptors on phagocytic cells, placentalcells, mast cells, etc. The light and heavy chains each have a variableregion and a constant region. Accordingly, the invention relates tohumanized immunoglobulins having binding specificity to B7-2 or B7-1.The humanized B7-1 or B7-2 immunoglobulin comprises a light chain and aheavy chain in which two light chains and two heavy chains form thetetramer.

The variable region further constitutes two types of regions, aframework region (FR) and a complementarity determining region (CDR).CDRs are hypervariable regions that contain most of the amino acidsequence variation in between immunoglobulins. Proceeding from theamino-terminus, these regions are designated CDR1, CDR2 and CDR3,respectively. See FIGS. 1A-1B, 2A-2B, 6A-6B and 7A-7B. The CDRs areconnected by more conserved FRs. Proceeding from the amino-terminus,these regions are designated FR1, FR2, FR3, and FR4, respectively. Thelocations of CDR and FR regions and a numbering system have been definedby Kabat et al. (Kabat, E. A. et al., Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, U.S. Government Printing Office (1991); Kabat, E. A.Structural Concepts in Immunology and Immunochemistry, Second Edition,Holt, Rinehart and Winston, New York (1976); Kabat, E. A. Sequences ofImmunoglobulin Chains: Tabulation and Analysis of Amino Acid Sequencesof Precursors, V-regions, C-regions, J-Chain and β2-Microglobulins, U.S.Department of Health, Education and Welfare, Public Health Service,(1979); Kabat, E. A. Structural Concepts in Immunology andImmunochemistry, Holt, Rinehart and Winston, New York (1968); Kabat, E.A. Experimental Immunochemistry, Second Edition, Springfield, Thomas(1967). During the process of humanizing an immunoglobulin, one or moreof the CDRs from an antibody having specificity for B7-2 or B7-1 from anon-human species is grafted into the FRs of a human antibody. Inaddition, certain non-human framework substitutes can be made accordingto the methods described herein. The resulting humanized antibody hasCDRs from a non-human species such as a mouse and FRs from a humanantibody, whereby the humanized antibody maintains its antigenicspecificity and affinity to B7-1 or B7-2.

The invention also relates to a B7-1 or B7-2 humanized immunoglobulinlight chain, or a B7-1 or B7-2 humanized immunoglobulin heavy chain. Inone embodiment, the invention relates to a humanized B7-2 light chaincomprising one or more light chain CDRs (e.g., CDR1 (SEQ ID NO: 16),CDR2 (SEQ ID NO: 18) and/or CDR3 (SEQ ID NO: 20)) of non-human originand a human light chain framework region (See FIG. 2B). In anotherembodiment, the invention relates to a humanized B7-2 immunoglobulinheavy chain comprising one or more heavy chain CDRs (e.g., CDR1 (SEQ IDNO: 10), CDR2 (SEQ ID NO: 12), and/or CDR3 (SEQ ID NO: 14)) of non-humanorigin and a human heavy chain framework region (See FIG. 2A). The CDRscan be derived from a non-human immunoglobulin such as murine heavy(e.g., SEQ ID NO: 1, FIG. 1A) and light (e.g., SEQ ID NO: 3, FIG. 1B)variable region chains of the 3D1 antibody which are specific to B7-2.

In another embodiment, the invention relates to a humanized B7-1 lightchain comprising one or more light chain CDRs (e.g., CDR1 (SEQ ID NO:36), CDR2 (SEQ ID NO: 38) and/or CDR3 (SEQ ID NO: 40)) of non-humanorigin and a human light chain framework region (See FIG. 7B). Theinvention also pertains to a B7-1 humanized immunoglobulin heavy chaincomprising one or more heavy chain CDRs (e.g., CDR1 (SEQ ID NO: 30),CDR2 (SEQ ID NO: 32), and/or CDR3 (SEQ ID NO: 34)) of non-human originand a human heavy chain framework region (See FIG. 7A). The CDRs can bederived from a non-human immunoglobulin such as murine heavy (e.g., SEQID NO: 21, FIG. 6A) and light (e.g., SEQ ID NO: 23, FIG. 6B) variableregion chains of the 1F1 antibody which are specific to B7-1.

The invention also embodies the humanized anti-B7-2 antibody expressedby a cell line deposited with the A.T.C.C., 10801 University Boulevard,Manassas, Va. 02110-2209, on May 5, 1998, A.T.C.C. No: CRL-12524. Thecell line which expresses the humanized anti-B7-2 antibody, depositedwith the A.T.C.C., is designated as a recombinant CHO cell line(PA-CHO-DUKX-1538) expressing the humanized anti-human B7-2 (CD86)monoclonal antibody (#HF2-3D1) of the IgG2.M3 isotype.

The invention embodies the humanized anti-B7-1 antibody expressed by acell line deposited with the A.T.C.C., 10801 University Boulevard,Manassas, Va. 02110-2209, on Jun. 22, 1999, under A.T.C.C. No: PTA-263.The cell line which expresses the humanized anti-B7-1 antibody,deposited with the A.T.C.C., is designated as a recombinant CHO cellline (PA-CHO-DUKX-1538) expressing the humanized anti-human B7-1 (CD80)monoclonal antibody (190 1F1).

Human immunoglobulins can be divided into classes and subclasses,depending on the isotype of the heavy chain. The classes include IgG,IgM, IgA, IgD and IgE, in which the heavy chains are of the gamma (γ),mu (μ), alpha (α), delta (δ) or epsilon (ε) type, respectively.Subclasses include IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, in which theheavy chains are of the γ1, γ2, γ3, γ4, α1 and α2 type, respectively.Human immunoglobulin molecules of a selected class or subclass maycontain either a kappa (κ) or lambda (λ) light chain. See e.g., Cellularand Molecular Immunology, Wonsiewicz, M. J., Ed., Chapter 45, pp. 41-50,W.B. Saunders Co, Philadelphia, Pa. (1991); Nisonoff, A., Introductionto Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, SinauerAssociates, Inc., Sunderland, Mass. (1984).

The terms “HF2.3D1” and “3D1” refer to a murine immunoglobulin specificto B7-2. The terms “humanized HF2.3D1,” “humanized 3D1”, “hu3D1,” “B7-2humanized immunoglobulin” or “humanized B7-2 immunoglobulin” refer to ahumanized immunoglobulin specific to human B7-2 (e.g., mouse anti-humanB7-2 antibody). The terms “1F1” or “mouse 1F1” refer to a murineimmunoglobulin that is specific to B7-1. The terms “humanized 1F1”,“hu1F1,” “B7-1 humanized immunoglobulin” or “humanized B7-1immunoglobulin” refer to a humanized immunoglobulin specific to humanB7-1 (e.g., mouse anti-human B7-1 antibody). The term “B7 molecules”refer to the B7-1 and B7-2 molecules. The term “B7 antibodies” refer tothe anti-human B7-1 and anti-human B7-2 antibodies.

The terms “immunoglobulin” or “antibody” include whole antibodies andbiologically functional fragments thereof. Such biologically functionalfragments retain at least one antigen binding function of acorresponding full-length antibody and preferably, retain the ability toinhibit the interaction of B7-2 or B7-1 with one or more of itsreceptors (e.g., CD28, CTLA4). In a preferred embodiment, biologicallyfunctional fragments can inhibit binding of B7-2 and/or B7-1 formanipulation of the co-stimulatory pathway. Examples of biologicallyfunctional antibody fragments which can be used include fragmentscapable of binding to B7-2 or B7-1, such as single chain antibodies, Fv,Fab, Fab′ and F(ab′)₂ fragments. Such fragments can be produced byenzymatic cleavage or by recombinant techniques. For instance, papain orpepsin cleavage can be used to generate Fab or F(ab′)₂ fragments,respectively. Antibodies can also be produced in a variety of truncatedforms using antibody genes in which one or more stop codons have beenintroduced upstream of the natural stop site. For example, a chimericgene encoding the heavy chain of a F(ab′)₂ fragment can be designed toinclude DNA sequences encoding the CH₁ , domain and hinge region of theheavy chain. The invention includes single chain antibodies (e.g., asingle chain FV) that contain both portions of the heavy and lightchains.

The term “humanized immunoglobulin” as used herein refers to animmunoglobulin comprising portions of immunoglobulins of differentorigin, wherein at least one portion is of human origin. For example,the humanized antibody can comprise portions derived from animmunoglobulin of non-human origin with the requisite specificity, suchas a mouse, and from immunoglobulin sequences of human origin (e.g.,chimeric immunoglobulin). These portions can be joined togetherchemically by conventional techniques (e.g., synthetic) or prepared as acontiguous polypeptide using genetic engineering techniques (e.g., DNAencoding the protein portions of the chimeric antibody can be expressedto produce a contiguous polypeptide chain). Another example of ahumanized immunoglobulin of the invention is an immunoglobulincontaining one or more immunoglobulin chains comprising a CDR derivedfrom an antibody of non-human origin and a framework region derived froma light and/or heavy chain of human origin (e.g., CDR-grafted antibodieswith or without framework changes). Chimeric or CDR-grafted single chainantibodies are also encompassed by the term humanized immunoglobulin.See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al.,European Patent No. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397;Boss et al., European Patent No. 0,120,694 B1; Neuberger, M. S. et al.,WO 86/01533; Neuberger, M. S. et al., European Patent No. 0,194,276 B1;Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0,239,400B1; Padlan, E. A. et al., European Patent Application No. 0,519,596 A1.See also, Ladner et al., U.S. Pat. No. 4,946,778; Huston, U.S. Pat. No.5,476,786; and Bird, R. E. et al., Science, 242: 423-426 (1988),regarding single chain antibodies.

As embodied in the exemplified antibody of the present invention, theterm “humanized immunoglobulin” also refers to an immunoglobulincomprising a human framework, at least one CDR from a non-humanantibody, and in which any constant region present is substantiallyidentical to a human immunoglobulin constant region, e.g., at leastabout 60-90%, preferably at least 95% identical. Hence, all parts of ahumanized immunoglobulin, except possibly the CDR's, are substantiallyidentical to corresponding parts of one or more native humanimmunoglobulin sequences. In some instances, the humanizedimmunoglobulin, in addition to CDRs from a non-human antibody, wouldinclude additional non-human residues in the human framework region.

The design of humanized immunoglobulins can be carried out as follows.When an amino acid falls under the following categories, the frameworkamino acid of a human immunoglobulin to be used (acceptorimmunoglobulin) is replaced by a framework amino acid from aCDR-providing non-human immunoglobulin (donor immunoglobulin):

-   -   (a) the amino acid in the human framework region of the acceptor        immunoglobulin is unusual for human immunoglobulin at that        position, whereas the corresponding amino acid in the donor        immunoglobulin is typical for human immunoglobulin in that        position:    -   (b) the position of the amino acid is immediately adjacent to        one of the CDR's; or    -   (c) the amino acid is capable of interacting with the CDR's in a        tertiary structure immunoglobulin model (see, Queen et al., op.        cit., and Co et al., Proc. Natl. Acad. Sci. USA 88, 2869        (1991)).

For a detailed description of the production of humanizedimmunoglobulins, See Queen et al., op. cit. and Co et al, op. cit. andU.S. Pat. Nos. 5,585,089; 5,693,762, 5,693,761, and 5,530,101.

Usually, the CDR regions in humanized antibodies are substantiallyidentical, and more usually, identical to the corresponding CDR regionsin the mouse antibody from which they were derived. Although not usuallydesirable, it is sometimes possible to make one or more conservativeamino acid substitutions of CDR residues without appreciably affectingthe binding affinity of the resulting humanized immunoglobulin.Occasionally, substitutions of CDR regions can enhance binding affinity.

Other than for the specific amino acid substitutions discussed above,the framework regions of humanized immunoglobulins are usuallysubstantially identical, and more usually, identical to the frameworkregions of the human antibodies from which they were derived. Of course,many of the amino acids in the framework region make little or no directcontribution to the specificity or affinity of an antibody. Thus, manyindividual conservative substitutions of framework residues can betolerated without appreciable change of the specificity or affinity ofthe resulting humanized immunoglobulin.

The antigen binding region of the humanized B7-2 immunoglobulin (thenon-human portion) can be derived from an immunoglobulin of non-humanorigin, referred to as a donor immunoglobulin, having specificity forB7-2 (e.g., the 3D1 antibody) or B7-1 (e.g., the 1F1 antibody). Forexample, a suitable antigen binding region for the humanized B7-2antibody can be derived from the HF2.3D1 monoclonal antibody, a murineanti-human B7-2 antibody. U.S. Ser. No. 08/101,624, filed on Jul. 26,1993, Ser. No. 08/109,393, filed Aug. 19, 1993 and 08/147,773, filedNov. 3, 1993, entitled, “B7-2:CTLA4/CD28 Counter Receptor”. See also,Freeman, et al., WO 95/03408, “B7-2: CTLA4/CD 28 Counter Receptor,published on Feb. 2, 1995. A suitable antigen binding region for thehumanized B7-1 antibody can be derived from the murine 1F1 monoclonalantibody, a murine-anti-human B7-1 antibody. Other sources include B7-2or B7-1 specific antibodies obtained from non-human sources, such asrodent (e.g., mouse and rat), rabbit, pig, goat or non-human primate(e.g., monkey) or camelid animals (e.g., camels and llamas).

Additionally, other polyclonal or monoclonal antibodies, such asantibodies which bind to the same or similar epitope as the murineHF2.3D1 or 1F1 antibodies, can be made (e.g., Kohler et al., Nature,256:495-497 (1975); Harlow et al., 1988, Antibodies: A LaboratoryManual, (Cold Spring Harbor, N.Y.); and Current Protocols in MolecularBiology, Vol. 2 (Supplement 27, Summer '94), Ausubel et al., Eds. (JohnWiley & Sons: New York, N.Y.), Chapter 11 (1991)). For example,antibodies can be raised against an appropriate immunogen in a suitablemammal such as a mouse, rat, rabbit, sheep, or camelid. Cells bearingB7-2 or B7-1, membrane fractions containing B7-2 or B7-1 immunogenicfragments of B7-2 or B7-1, and a B7-2 or B7-1 peptide conjugated to asuitable carrier are examples of suitable immunogens (e.g., DNA orpeptide immunogens). Antibody-producing cells (e.g., a lymphocyte) canbe isolated, for example, from the lymph nodes or spleen of an immunizedanimal. The cells can then be fused to a suitable immortalized cell(e.g., a myeloma cell line), thereby forming a hybridoma. Fused cellscan be isolated employing selective culturing techniques. Cells whichproduce antibodies with the desired specificity can be selected by asuitable assay, such as an ELISA. Immunoglobulins of non-human originhaving binding specificity for B7-2 or B7-1 can also be obtained fromantibody libraries, such as a phage library comprising non-human Fabmolecules. Humanized immunoglobulins can be made using other techniques.

In one embodiment, the antigen binding region of the humanizedimmunoglobulins comprise a CDR of non-human origin. In this embodiment,humanized immunoglobulins having binding specificity for B7-2 or B7-1comprise at least one CDR of non-human origin. For example, CDRs can bederived from the light and heavy chain variable regions ofimmunoglobulins of non-human origin, such that a humanized B7-2immunoglobulin includes substantially the heavy chain CDR1 (e.g., SEQ IDNO: 10), CDR2 (e.g., SEQ ID NO: 12) and/or CDR3 (e.g., SEQ ID NO: 14)amino acid sequences, and/or light chain CDR1 (e.g., SEQ ID NO: 16),CDR2 (e.g., SEQ ID NO: 18) and/or CDR3 (e.g., SEQ ID NO: 20) amino acidsequences, from one or more immunoglobulins of non-human origin, and theresulting humanized immunoglobulin has binding specificity for B7-2.CDRs can also be derived from light and heavy chain variable regions ofimmunoglobulins of non-human origin that are specific for B7-1. Thehumanized B7-1 antibody comprises substantially the heavy chain CDR1(SEQ ID NO: 30), CDR2 (SEQ ID NO: 32) and/or CDR3 (e.g., SEQ ID NO: 34)amino acid sequences, and/or light chain CDR1 (SEQ ID NO: 36), CDR2 (SEQID NO: 38) and/or CDR3 (SEQ ID NO: 40) amino acid sequences, from one ormore immunoglobulins of non-human origin, and the resulting humanizedimmunoglobulin has binding specificity for B7-1. All three CDRs of aselected chain can be substantially the same as the CDRs of thecorresponding chain of a donor, and preferably, all three CDRs of thelight and heavy chains are substantially the same as the CDRs of thecorresponding donor chain. The nucleic acid sequences of the B7-2 heavychain CDR1 (e.g., SEQ ID NO: 9), CDR2 (e.g., SEQ ID NO: 11) and CDR3(e.g., SEQ ID NO: 13) and/or B7-2 light chain CDR1 (e.g., SEQ ID NO:15), CDR2 (e.g., SEQ ID NO: 17), and CDR3 (e.g., SEQ ID NO: 19) can alsobe used in grafting the CDRs into the human framework. Additionally, thenucleic acid sequences of the B7-1 heavy chain CDR1 (SEQ ID NO: 29),CDR2 (SEQ ID NO: 31) and CDR3 (SEQ ID NO: 33) and/or B7-1 light chainCDR1 (SEQ ID NO: 35), CDR2 (SEQ ID NO: 37) and CDR3 (SEQ ID NO:39) canbe used in grafting the CDRs into the human framework.

In another embodiment, the invention pertains to humanizedimmunoglobulins having a binding specificity for either B7-2 or B7-1comprising a heavy chain and a light chain. The light chain can comprisea CDR derived from an antibody of non-human origin which binds B7-2 orB7-1 and a FR derived from a light chain of human origin. For example,the light chain can comprise CDR1, CDR2 and/or CDR3 which have the aminoacid sequence set forth below or an amino acid sequence substantiallythe same as the amino acid sequence such that the antibody specificallybinds to B7-2: CDR1 KSSQSLLNSRTRENYLA (SEQ ID NO: 16), CDR2 WASTRES (SEQID NO: 18), and CDR3 TQSYNLYT (SEQ ID NO: 20). The heavy chain cancomprise a CDR derived from an antibody of non-human origin which bindsB7-2 and a FR derived from a heavy chain of human origin. For example,the B7-2 heavy chain can comprise CDR1, CDR2 and CDR3 which have theamino acid sequence set forth below or an amino acid sequencesubstantially the sane as said amino acid sequence such that theantibody specifically binds to the B7-2: heavy chain: CDR1 DYAIQ (SEQ IDNO: 10), CDR2 VINIYYDNTNYNQKFKG (SEQ ID NO: 12), CDR3 AAWYMDY (SEQ IDNO: 14).

The light chain that is specific to B7-1 can comprise CDR1, CDR2 and/orCDR3 that have the amino acid sequence set forth below or an amino acidsequence substantially the same as the amino acid sequence such that theantibody specifically binds to B7-1: CDR1 SVSSSISSSNLH (SEQ ID NO: 30),CDR2 GTSNLAS (SEQ ID NO: 32) and CDR3 QQWSSYPLT (SEQ ID NO: 34). Theheavy chain can comprise a CDR derived from an antibody of non-humanorigin which binds B7-1 and a FR derived from a heavy chain of humanorigin. The heavy chain that is specific to B7-1 can comprise CDR1, CDR2and/or CDR3 that have the amino acid sequence set forth below or anamino acid sequence substantially the same as the amino acid sequencesuch that the antibody specifically binds to B7-1: CDR1 DYYMH (SEQ IDNO: 36), CDR2 WIDPENGNTLYDPKFQG (SEQ ID NO: 38), and CDR3 EGLFFAY (SEQID NO: 40).

An embodiment of the invention is a humanized immunoglobulin whichspecifically binds to B7-2 and comprises a humanized light chaincomprising three light chain CDRs from the mouse 3D1 antibody and alight chain variable region framework sequence from a humanimmunoglobulin light chain. The invention further comprises a B7-2humanized heavy chain that comprises three heavy chain CDRs from themouse 3D1 antibody and a heavy chain variable region framework sequencefrom a human immunoglobulin heavy chain. The mouse 3D1 antibody canfurther have a mature light chain variable domain as shown in FIG. 1B(SEQ ID NO.: 4) and a mature heavy chain variable domain as shown inFIG. 1A (SEQ ID NO.: 2).

In another embodiment of the invention is a humanized immunoglobulinwhich specifically binds to B7-1 and comprises a humanized light chaincomprising three light chain CDRs from the mouse 1F1 antibody, and alight chain variable region framework sequence from a humanimmunoglobulin light chain. The invention further comprises a B7-1humanized heavy chain that comprises three heavy chain CDRs from themouse 1F1 antibody, respectively, and a heavy chain variable regionframework sequence from a human immunoglobulin heavy chain. The mouse1F1 antibody can have a mature light chain variable domain as shown inFIG. 6B (SEQ ID NO: 24) and a mature heavy chain variable domain, asshown in FIG. 6A (SEQ ID NO: 22).

The portion of the humanized immunoglobulin or immunoglobulin chainwhich is of human origin (the human portion) can be derived from anysuitable human immunoglobulin or immunoglobulin chain. For example, ahuman constant region or portion thereof, if present, can be derivedfrom the κ or λ light chains, and/or the γ (e.g., γ1, γ2, γ3, γ4), μ, α(e.g., α1, α2), δ or ε heavy chains of human antibodies, includingallelic variants. A particular constant region, such as IgG2 or IgG4,variants or portions thereof can be selected to tailor effectorfunction. For example, a mutated constant region, also referred to as a“variant,” can be incorporated into a fusion protein to minimize bindingto Fc receptors and/or ability to fix complement (see e.g., Winter etal., U.S. Pat. Nos. 5,648,260 and 5,624,821; GB 2,209,757 B; Morrison etal., WO 89/07142; Morgan et al., WO 94/29351, Dec. 22, 1994). Inaddition, a mutated IgG2 Fc domain can be created that reduces themitogenic response, as compared to natural Fc regions (see e.g., Tso etal., U.S. Pat. No. 5,834,597, the teachings of which are incorporated byreference herein in their entirety). See Example 3 for mutationsperformed to the humanized anti-B7-2 antibody and Example 10 formutations performed to the humanized anti-B7-1 antibody.

If present, human FRs are preferably derived from a human antibodyvariable region having sequence similarity to the analogous orequivalent region of the antigen binding region donor. Other sources ofFRs for portions of human origin of a humanized immunoglobulin includehuman variable consensus sequences (See, Kettleborough, C. A. et al.,Protein Engineering 4:773-783 (1991); Queen et al., U.S. Pat. Nos.5,585,089, 5,693,762 and 5,693,761). For example, the sequence of theantibody or variable region used to obtain the non-human portion can becompared to human sequences as described in Kabat, E. A., et al.,Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, U.S. Government Printing Office(1991). In a preferred embodiment, the FRs of the humanizedimmunoglobulin chains are derived from a human variable region having atleast about 60% overall sequence identity, and preferably at least about80% overall sequence identity, with the variable region of the non-humandonor (e.g., murine HF2.3D1 or 1F1 antibody). For example, the overallsequence identity between the mouse HF2.3D1 and human H₂F light chainvariable framework regions is 82.5%, and the overall sequence identitybetween the mouse HF2.3D1 and human III-2R heavy chain variableframework regions is 62.5%. For the B7-1 antibody, the overall sequenceidentity between the murine 1F1 and humanized III-2R light chainvariable frame-work region is 69%, and the overall sequence identitybetween the murine III-2R heavy chain variable frame-work region is 79%.

The phrase “substantially identical,” in context of two nucleic acids orpolypeptides (e.g., DNAs encoding a humanized immunoglobulin or theamino acid sequence of the humanized immunoglobulin) refers to two ormore sequences or subsequences that have at least about 80%, mostpreferably 90-95% or higher nucleotide or amino acid residue identity,when compared and aligned for maximum correspondence, as measured usingthe following sequence comparison method and/or by visual inspection.Such “substantially identical” sequences are typically considered to behomologous. Preferably, the “substantial identity” exists over a regionof the sequences that is at least about 50 residues in length, morepreferably over a region of at least about 100 residues, and mostpreferably the sequences are substantially identical over at least about150 residues, or over the full length of the two sequences to becompared. As described below, any two antibody sequences can only bealigned in one way, by using the numbering scheme in Kabat. Therefore,for antibodies, percent identity has a unique and well-defined meaning.

Amino acids from the variable regions of the mature heavy and lightchains of immunoglobulins are designated Hx and Lx respectively, where xis a number designating the position of an amino acid according to thescheme of Kabat, Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md., 1987 and 1991). Kabatlists many amino acid sequences for antibodies for each subgroup, andlists the most commonly occurring amino acid for each residue positionin that subgroup. Kabat uses a method for assigning a residue number toeach amino acid in a listed sequence. Kabat's scheme is extendible toother antibodies not included in the compendium by aligning the antibodyin question with one of the consensus sequences in Kabat. The use of theKabat numbering system readily identifies amino acids at equivalentpositions in different antibodies. For example, an amino acid at the L50position of a human antibody occupies the equivalent position to anamino acid position L50 of a mouse antibody.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Thevariable regions of each light/heavy chain pair form the antibodybinding site. Thus, an intact antibody has two binding sites.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Fundamental Immunology, Paul, W., ed., 3rd ed. Raven Press, N.Y., 1993,Ch. 9).

From N-terminal to C-terminal, both light and heavy chain variableregions comprise alternating framework and (CDRs)” FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each region is inaccordance with the definitions of Kabat (1987) and (1991), supra and/orChothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature342:878-883 (1989).

In one embodiment, the humanized immunoglobulins comprise at least oneof the FRs derived from one or more chains of an antibody of humanorigin. Thus, the FR can include a FR1, FR2, FR3 and/or FR4 derived fromone or more antibodies of human origin. Preferably, the human portion ofa selected humanized chain includes FR1, FR2, FR3 and/or FR4 derivedfrom a variable region of human origin (e.g., from a humanimmunoglobulin chain, from a human consensus sequence). In a preferredembodiment, the FRs for the B7-2 light chain variable region are fromthe H₂F human antibody and the FRs for the B7-2 heavy chain variableregion are from the I2R human antibody. The FRs for B7-1 heavy and lightchain variable regions are from the 1-2R antibody.

The immunoglobulin portions of non-human and human origin for use in theinvention have sequences that are identical to immunoglobulins orimmunoglobulin portions from which they are derived, or to variantsthereof. Such variants include mutants differing by the addition,deletion, or substitution of one or more residues. As indicated above,the CDRs which are of non-human origin are substantially the same as inthe non-human donor, and preferably are identical to the CDRs of thenon-human donor. As described herein, changes in the FR, such as thosewhich substitute a residue of the FR of human origin with a residue fromthe corresponding position of the donor can be made. One or moremutations in the FR can be made, including deletions, insertions andsubstitutions of one or more amino acids. Several such substitutions aredescribed in the design of a humanized HF2.3D1 antibody in Example 2 andfor the humanized 1F1 in Example 9. For a selected humanized antibody orchain, framework mutations can be designed as described herein.Preferably, the B7-2 and B7-1 humanized immunoglobulins can bind B7-2and B7-1, respectively, with an affinity similar to or better than thatof the non-human donor. Variants can be produced by a variety ofsuitable methods, including mutagenesis of non-human donor or acceptorhuman chains.

The humanized immunoglobulins of the invention have binding specificityfor human B7-2 or B7-1, and include humanized immunoglobulins (includingfragments) which can bind determinants of B7-2 or B7-1. In a preferredembodiment, the humanized immunoglobulin of the present invention has atleast one functional characteristic of murine HF2.3D1 or 1F1 antibody,such as binding function (e.g., having specificity for B7-2 or B7-1,having the same or similar epitopic specificity), and/or inhibitoryfunction (e.g., the ability to inhibit the binding of a cell bearingCD28 or CTLA4 to the B7-2 or B7-1 ligand). Thus, preferred humanizedimmunoglobulins can have the binding specificity of the murine HF2.3D1or 1F1 antibody, the epitopic specificity of the murine HF2.3D1 or 1F1antibody (e.g., can compete with murine HF2.3D1 or 1F1, a chimericHF2.3D1 or 1F1 antibody, or humanized HF2.3D1 or 1F1 for binding to B7-2or B7-1, respectively) and/or inhibitory function.

The binding function of a humanized immunoglobulin having bindingspecificity for B7-2 or B7-1 can be detected by standard immunologicalmethods, for example, using assays which monitor formation of a complexbetween humanized immunoglobulin and B7-2 or B7-1 (e.g., a membranefraction comprising B7-2 or B7-1, or human lymphocyte cell line orrecombinant host cell comprising nucleic acid which expresses B7-2 orB7-1).

Binding and/or adhesion assays or other suitable methods can also beused in procedures for the identification and/or isolation of humanizedimmunoglobulins (e.g., from a library) with the requisite specificity(e.g., an assay which monitors adhesion between a cell bearing a B7-2 orB7-1 receptor and B7 molecule, or other suitable methods).

The immunoglobulin portions of non-human and human origin for use in theinvention include light chains, heavy chains and portions of light andheavy chains. These immunoglobulin portions can be obtained or derivedfrom immunoglobulins (e.g., by de novo synthesis of a portion), ornucleic acids encoding an immunoglobulin or chain thereof having thedesired property (e.g., binds B7-2 or B7-1, sequence similarity) can beproduced and expressed. Humanized immunoglobulins comprising the desiredportions (e.g., antigen binding region, CDR, FR, C region) of human andnon-human origin can be produced using synthetic and/or recombinantnucleic acids to prepare genes (e.g., cDNA) encoding the desiredhumanized chain. To prepare a portion of a chain, one or more stopcodons can be introduced at the desired position. For example, nucleicacid sequences coding for newly designed humanized variable regions canbe constructed using PCR mutagenesis methods to alter existing DNAsequences (see e.g., Kamman, M., et al., Nucl. Acids Res. 17:5404(1989)). PCR primers coding for the new CDRs can be hybridized to a DNAtemplate of a previously humanized variable region which is based on thesame, or a very similar, human variable region (Sato, K., et al., CancerResearch 53:851-856 (1993)). If a similar DNA sequence is not availablefor use as a template, a nucleic acid comprising a sequence encoding avariable region sequence can be constructed from syntheticoligonucleotides (see e.g., Kolbinger, F., Protein Engineering 8:971-980(1993)). A sequence encoding a signal peptide can also be incorporatedinto the nucleic acid (e.g., on synthesis, upon insertion into avector). If the natural signal peptide sequence is unavailable, a signalpeptide sequence from another antibody can be used (see, e.g.,Kettleborough, C. A., Protein Engineering 4:773-783 (1991)). Using thesemethods, methods described herein or other suitable methods, variantscan be readily produced. In one embodiment, cloned variable regions canbe mutagenized, and sequences encoding variants with the desiredspecificity can be selected (e.g., from a phage library; see e.g.,Krebber et al., U.S. Pat. No. 5,514,548; Hoogengoom et al., WO 93/06213,published Apr. 1, 1993)).

Nucleic Acids and Constructs Comprising Same:

The invention also relates to isolated and/or recombinant (including,e.g., essentially pure) nucleic acids comprising sequences which encodea humanized B7-1 or B7-2 immunoglobulin, or humanized B7-1 or B7-2immunoglobulin light or heavy chain of the present invention.

Nucleic acids referred to herein as “isolated” are nucleic acids whichhave been separated away from the nucleic acids of the genomic DNA orcellular RNA of their source of origin (e.g., as it exists in cells orin a mixture of nucleic acids such as a library), and include nucleicacids obtained by methods described herein or other suitable methods,including essentially pure nucleic acids, nucleic acids produced bychemical synthesis, by combinations of biological and chemical methods,and recombinant nucleic acids which are isolated (see e.g., Daugherty,B. L. et al., Nucleic Acids Res., 19(9): 2471-2476 (1991); Lewis, A. P.and J. S. Crowe, Gene, 101: 297-302 (1991)).

Nucleic acids referred to herein as “recombinant” are nucleic acidswhich have been produced by recombinant DNA methodology, including thosenucleic acids that are generated by procedures which rely upon a methodof artificial recombination, such as the polymerase chain reaction (PCR)and/or cloning into a vector (e.g., plasmid) using restriction enzymes.“Recombinant” nucleic acids are also those that result fromrecombination events that occur through the natural mechanisms of cells,but are selected for after the introduction to the cells of nucleicacids designed to allow and make probable a desired recombination event.

The invention also relates, more specifically, to isolated and/orrecombinant nucleic acids comprising a nucleotide sequence which encodesa humanized HF2.3D1 or 1F1 immunoglobulin, also referred to as“humanized 3D1” or “humanized 1F1,” respectively, (e.g., a humanizedimmunoglobulin of the invention in which the non-human portion isderived from the murine HF2.3D1 or 1F1 monoclonal antibody), or chainthereof. In one embodiment, the light chain comprises threecomplementarity determining regions derived from the light chain of theHF2.3D1 or 1F1 antibody, and the heavy chain comprises threecomplementarity determining regions derived from the heavy chain of theHF2.3D1 or 1F1 antibody. Such nucleic acids include, for example, (a) anucleic acid comprising a sequence which encodes a polypeptidecomprising the amino acid sequence of the heavy chain variable region ofa humanized HF2.3D1 or 1F1 immunoglobulin (e.g., SEQ ID NO: 5, See FIG.2A or SEQ ID NO: 25, See FIG. 7A), (b) a nucleic acid comprising asequence which encodes a polypeptide comprising the amino acid sequenceof the light chain variable region of a humanized HF2.3D1 or 1F1immunoglobulin (e.g., SEQ ID NO: 7, See FIG. 2B or SEQ ID NO: 27, SeeFIG. 7B), (c) a nucleic acid comprising a sequence which encodes atleast a functional portion of the light or heavy chain variable regionof a humanized HF2.3D1 or 1F1 immunoglobulin (e.g., a portion sufficientfor antigen binding of a humanized immunoglobulin which comprises thechain). Due to the degeneracy of the genetic code, a variety of nucleicacids can be made which encode a selected polypeptide. In oneembodiment, the nucleic acid comprises the nucleotide sequence of thevariable region as set forth or substantially as set forth in FIG. 2Aand/or FIG. 2B, or FIG. 7A and/or FIG. 7B, including double orsingle-stranded polynucleotides. Isolated and/or recombinant nucleicacids meeting these criteria can comprise nucleic acids encodingsequences identical to sequences of humanized HF2.3D1 or humanized 1F1antibody or variants thereof, as discussed above.

Nucleic acids of the invention can be used in the production ofhumanized immunoglobulins having binding specificity for B7-2 or B7-1.For example, a nucleic acid (e.g., DNA) encoding a humanizedimmunoglobulin of the invention can be incorporated into a suitableconstruct (e.g., a vector) for further manipulation of sequences or forproduction of the encoded polypeptide in suitable host cells.

Method of Producing Humanized Immunoglobulins Having Specificity forB7-2 and/or B7-1:

Another aspect of the invention relates to a method of preparing ahumanized immunoglobulin which has binding specificity for B7-2 or B7-1.The humanized immunoglobulin can be obtained, for example, by theexpression of one or more recombinant nucleic acids encoding a humanizedimmunoglobulin having binding specificity for B7-2 or B7-1 in a suitablehost cell.

Constructs or expression vectors suitable for the expression of ahumanized immunoglobulin having binding specificity for B7-2 or B7-1 arealso provided. The constructs can be introduced into a suitable hostcell, and cells which express a humanized immunoglobulin of theinvention, can be produced and maintained in culture. Suitable hostcells can be procaryotic, including bacterial cells such as E. coli, B.subtilis and or other suitable bacteria, or eucaryotic, such as fungalor yeast cells (e.g., Pichia pastoris, Aspergillus species,Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa),or other lower eucaryotic cells, and cells of higher eucaryotes such asthose from insects (e.g., Sf9 insect cells (WO 94/26087, O'Connor,published Nov. 24, 1994)). Suitable host cells can also come fromplants, transgenic animals, or mammals (e.g., COS cells, NSO cells,SP2/0, Chinese hamster ovary cells (CHO), HuT 78 cells, 293 cells).(See, e.g., Ausubel, F. M. et al., eds. Current Protocols in MolecularBiology, Greene Publishing Associates and John Wiley & Sons Inc.,(1993)).

Host cells which produce a humanized immunoglobulin having bindingspecificity for B7-2 or B7-1 can be produced as follows. For example, anucleic acid encoding all or part of the coding sequence for the desiredhumanized immunoglobulin can be inserted into a nucleic acid vector,e.g., a DNA vector, such as a plasmid, virus or other suitableexpression unit. A variety of vectors are available, including vectorswhich are maintained in single copy or multiple copy, or which becomeintegrated into the host cell chromosome.

Suitable expression vectors can contain a number of components,including, but not limited to one or more of the following: an origin ofreplication; a selectable marker gene; one or more expression controlelements, such as a transcriptional control element (e.g., a promoter,an enhancer, terminator), and/or one or more translation signals; asignal sequence or leader sequence for membrane targeting or secretion.In a construct, a signal sequence can be provided by the vector or othersource. For example, the transcriptional and/or translational signals ofan immunoglobulin can be used to direct expression.

A promoter can be provided for expression in a suitable host cell.Promoters can be constitutive or inducible. For example, a promoter canbe operably linked to a nucleic acid encoding a humanized immunoglobulinor immunoglobulin chain, such that it directs expression of the encodedpolypeptide. A variety of suitable promoters for procaryotic (e.g., lac,tac, T3, and T7 promoters for E. coli) and eucaryotic (e.g., yeastalcohol dehydrogenase (ADH) and SV40, CMV) hosts are available.

In addition, the expression vectors typically comprise a selectablemarker for selection of host cells carrying the vector, and, in the caseof replicable expression vector, an origin of replication. Genesencoding products which confer antibiotic or drug resistance are commonselectable markers and can be used in procaryotic (e.g., α-lactamasegene (ampicillin resistance) and Tet gene (tetracycline resistance)) andeucaryotic cells (e.g., neomycin (G418 or geneticin), gpt (mycophenolicacid), ampicillin, and hygromycin resistance genes). Dihydrofolatereductase marker genes permit selection with methotrexate in a varietyof hosts. Genes encoding the gene product of auxotrophic markers of thehost (e.g., LEU2, URA3 and HIS3) are often used as selectable markers inyeast. Use of viral (e.g., baculovirus) or phage vectors, and vectorswhich are capable of integrating into the genome of the host cell, suchas retroviral vectors, are also contemplated. The invention also relatesto cells carrying these expression vectors.

For example, a nucleic acid (e.g., one or more nucleic acids) encodingthe heavy and light chains of a humanized immunoglobulin having bindingspecificity for B7-2 or B7-1, or a construct (e.g., one or moreconstructs) comprising such nucleic acid(s), can be introduced into asuitable host cell by a method appropriate to the host cell selected(e.g., transformation, transfection, electroporation, infection), suchthat the nucleic acid(s) are operably linked to one or more expressioncontrol elements (e.g., in a vector, in a construct created by processesin the cell, integrated into the host cell genome). Host cells can bemaintained under conditions suitable for expression (e.g., in thepresence of inducer, suitable media supplemented with appropriate salts,growth factors, antibiotic, nutritional supplements, etc.), whereby theencoded polypeptide(s) are produced. If desired, the encoded protein(e.g., humanized HF2.3D1 or 1F1 antibody) can be isolated from, forexample, host cells, medium or milk. This process encompasses expressionin a host cell of a transgenic animal (see e.g., WO 92/03918, GenPharmInternational, published Mar. 19, 1992).

Fusion proteins can be produced in which a humanized immunoglobulin orimmunoglobulin chain is linked to a non-immunoglobulin moiety (e.g., amoiety which does not occur in immunoglobulins as found in nature) in anN-terminal location, C-terminal location or internal to the fusionprotein. For example, some embodiments can be produced by the insertionof a nucleic acid encoding immunoglobulin sequences into a suitableexpression vector, such as a pET vector (e.g., pET-15b, Novagen), aphage vector (e.g., pCANTAB 5 E, Pharmacia), or other vector (e.g.,pRIT2T Protein A fusion vector, Pharmacia). The resulting construct canbe introduced into a suitable host cell for expression. Upon expression,some fusion proteins can be isolated or purified from a cell lysate bymeans of a suitable affinity matrix (see e.g., Current Protocols inMolecular Biology (Ausubel, F. M. et al., eds., Vol. 2, Suppl. 26, pp.16.4.1-16.7.8 (1991)).

Therapeutic Methods and Compositions:

Two types of T-cells exist: helper T cells and cytotoxic T cells. Thehelper T cells can recognize an antigen that is coupled with a majorhistocompatibility complex (MHC). Antigen presenting cells internalizean antigen and re-express the antigen with the MHC molecule. Uponrecognition of the antigen, secretion of cytokines occur. Cytokinesecretion activates B-lymphocytes, cytotoxic T cells, phagocytes andother cells. However, cytokine secretion and cellular proliferationrequire more than recognition of the antigen. Complete T-cell activationrequires a second signal referred to as the “co-stimulatory signal.”These co-stimulatory signals serve to initiate, maintain, and regulatethe activation cascade. An important co-stimulatory pathway is calledthe B7:CD28/CTLA4 pathway.

The B7:CD28/CTLA4 pathway involves two co-stimulatory ligands, B7-1(CD80) and B7-2 (CD86). The B7-1 and B7-2 ligands which are present onthe antigen presenting cell each bind to two receptors on T-cells calledCD28 and CTLA4.

The expression of B7 polypeptides, B7-1 (CD80) and B7-2 (CD86), istightly regulated. (Linsley, P S et al., Immunity 1:793-801 (1994).Unstimulated antigen-presenting cells generally do not express B7-1 andB7-2, except in dendritic cells. After activation, dendritic cells,epidermal Langerhans' cells, B cells, and macrophages up-regulate theexpression of B7-2 and B7-1. Additionally, B7-2 can be expressed ongranulocytes and on T-cell molecules, and B7-1 is expressed infibroblasts and T-cell molecules. (Reiser, et al., New England J. ofMed., 335:18, 1369-1377, 1371 (1996).

In most immune responses, B7-2 is induced earlier than B7-1 and rises tohigher levels. B7-2 also affects the production of interleukin-4 (IL-4)and the generation of type 2 helper cells. B7 molecules (B7-1 and B7-2)are also responsible for costimulating CD8 T cells in the absence of CD4T cells which can be helpful in generating vaccines against melanoma. B7molecules can costimulate natural killer cells and γ/δ T cells. Hence,modulation of B7 molecules is helpful in anti-tumor and anti-microbialimmunity.

The B7:CD28/CTLA4 pathway participates in various disease statesincluding the pathogenesis of infectious diseases, asthma, autoimmunediseases, inflammatory disorders, the rejection of grafted organs andgraft versus host disease. This pathway also participates in prophylaxisand mechanisms that stimulate the immune system. Transfection with genesencoding costimulators, such as B7, are applicable for anti-tumor andanti-viral vaccines. Also, the B7 molecules participate in autoimmunediseases such as systemic lupus erythematosus, diabetes mellitus,insulitis, arthritis, inflammatory bowel disease, inflammatorydermatitis (psoriasis vulgaris and atopic dermatitis), and multiplesclerosis. (Reiser, et al., New England J. of Med., 335 (18): 1369(1996).

Accordingly, the invention encompasses methods for treating the disease,as described herein, comprising administering immunoglobulin(s) thatbinds to B7-1 and/or B7-2. The immunoglobulin should be administered intherapeutically effective amounts and, optionally, in a carrier. Inaddition to the diseases described herein, the immunoglobulin that bindB7-1 and/or B7-2 can be administered to a person having transplantedtissue, organ or cells. Inhibiting the B7 pathway prevents or reducesthe rejection of the transplanted tissue, organ or cell. The inventionpertains to treating acute and/or chronic transplant rejection for aprolonged period of time (e.g., days, months, years).

Therefore, modulating or influencing the B7 molecules role can be usefulin treating individuals with these diseases. B7 modulation is alsouseful in treating individuals with immune-related or autoimmunediseases and disorders in which B7-2 and/or B7-1 participates. Themodulation of B7-2 or B7-1 can also be used for diseases related to oraffected by IL-4 and/or the generation of type 2 helper cells. Thesedisorders/diseases can be treated using an antibody specific to B7-2and/or B7-1. Preferably, the antibody is a humanized antibody specificto B7-2 or B7-1. Treatment of these diseases can be facilitated withco-administration of an anti-B7-2 antibody, an anti-B7-1 antibody,including chimeric and humanized versions thereof, and/or antibodies tothe corresponding receptors, CD28 and CTLA4. Methods of treatment alsoinvolve co-administration of a humanized anti-B7-2 antibody or humanizedanti-B7-1 antibody with other standard therapy drugs (e.g., a drug thatis used to modulate the immune response of an individual having atransplanted organ, tissue, cell or the like), such as methotrexate,rapamycin, cyclosporin, steroids, anti-CD40 antibodies, and analogsthereof.

The invention includes methods for transplanting cells (e.g., bloodcells or components, or bone marrow) to an individual in need thereof.An individual in need thereof is one, for example, having a disease thatis treated with such a transplant (e.g., proliferative diseases such asleukemia, lymphoma, cancer; anemia such as sickle-cell anemia,thalassemia, and aplastic anemia; and myeloid dysplasia syndrome). Themethod comprises obtaining cells from a donor. Generally, donor bonemarrow contains both immature and mature lymphocytes. The blood cellsfrom a donor can be stem cells or immature blood cells in addition tobone marrow cells. The cells of the donor preferably comes from, but isnot limited to a person who has similar characteristics as thepatient/recipient (e.g., the donor's bone marrow is a match to thepatient's bone marrow). The characteristics that are analyzed todetermine whether a donor is a match to the patient are MHC class 1 and2 (e.g., HLA-A, HLA-B, and/or HLA-DR). The method involves contactingthe cells (e.g., bone marrow or other blood components) with animmunoglobulin specific to B7-1 and/or an immunoglobulin specific toB7-2 and recipient cells (e.g., lymphocyte from the patient) to obtain amixture, referred to as “treated cells”. The donor cells,immunoglobulin(s) and recipient cells are in contact for a period oftime sufficient for tolerance induction (e.g., about 1-48 hours,preferably about 36 hours). Tolerance induction (e.g., anergy) refers tothe lack of responsiveness to an antigen that has been induced with atreatment with B7-1 and/or B7-2 antibodies, such that the T-cell can nolonger adequately or fully respond to that antigen. Example 17. Therecipient cells (e.g., Peripheral Blood Lymphocytes (PBL), orlymphocytes that express class I antigens (MHC-I)) are irradiated toprevent cells from dividing. A substitute for recipient cells can betissue, organs or engineered cells that express MHC class I antigens,and B7-1 and/or B7-2 molecules. The method then includes introducing themixture (e.g., the treated cells) or treated bone marrow to the patient.This method of treatment is aimed at preventing graft vs. host disease.For example, cells in the treated bone marrow become tolerant torecipient alloantigen thereby reducing or eliminating graft vs. hostdisease. Accordingly, the methods of the present invention includetreating, preventing or aiding in the prevention of graft vs. hostdisease. The anti-B7-1 and anti-B7-2 antibodies reduce rejection bydonor bone marrow or donor cells of the recipient. However, the methodsare able to reduce rejection without significantly compromising thepatient's ability to detect and develop an immune response to otherforeign cells and antigens. Hence, these methods allow thetransplantation to be recipient specific, and allow for the rejection offoreign antigens without compromising the transplant. SeeExemplification Section.

The invention pertains to a pharmaceutical composition comprising ahumanized anti-B7-1 and/or humanized anti-B7-2 antibody, with or withouta carrier. A preferred embodiment is to administer the B7-1 and/or B7-2immunoglobulins in a tablet, injectable, or capsule form. In particular,the injectable form can be intravenous or subcutaneous injections. Theterms “pharmaceutically acceptable carrier” or a “carrier” refer to anygenerally acceptable excipient or drug delivery composition that isrelatively inert and non-toxic. Exemplary carriers include calciumcarbonate, sucrose, dextrose, mannose, albumin, starch, cellulose,silica gel, polyethylene glycol (PEG), dried skim milk, rice flour,magnesium stearate, and the like. Suitable formulations and additionalcarriers are described in Remington's Pharmaceutical Sciences, (17thEd., Mack Pub. Co., Easton, Pa.).

Suitable carriers (e.g., pharmaceutical carriers) also include, but arenot limited to sterile water, salt solutions (such as Ringer'ssolution), alcohols, polyethylene glycols, gelatin, carbohydrates suchas lactose, amylose or starch, magnesium stearate, talc, silicic acid,viscous paraffin, fatty acid esters, hydroxymethylcellulose, polyvinylpyrolidone, etc. Such preparations can be sterilized and, if desired,mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, coloring, and/or aromatic substances and the likewhich do not deleteriously react with the immunoglobulin. They can alsobe combined where desired with other active substances, e.g., enzymeinhibitors, to reduce metabolic degradation. A carrier (e.g., apharmaceutically acceptable carrier) is preferred, but not necessary toadminister the immunoglobulins.

For parenteral application, particularly suitable are injectable,sterile solutions, preferably oily or aqueous solutions, as well assuspensions, emulsions, or implants, including suppositories. Inparticular, carriers for parenteral administration include aqueoussolutions of dextrose, saline, pure water, ethanol, glycerol, propyleneglycol, peanut oil, sesame oil, polyoxyethylene-polyoxypropylene blockpolymers, and the like. Ampules, vials and syringes are convenient unitdosages.

Immunoglobulins of the invention can be administered intravenously,parenterally, intramuscular, subcutaneously, orally, nasally, byinhalation, by implant, by injection, or by suppository. The compositioncan be administered in a single dose or in more than one dose over aperiod of time to confer the desired effect.

The actual effective amounts of immunoglobulin can vary according to thespecific immunoglobulin being utilized, the particular compositionformulated, the mode of administration, and the age, weight, conditionof the patient, and severity of the disorder or disease, for example. Asused herein, an effective amount of the B7-2 and/or B7-2 immunoglobulinsis an amount which modulates or inhibits B7 molecules. Dosages for aparticular patient can be determined by one of ordinary skill in the artusing conventional considerations, (e.g. by means of an appropriate,conventional pharmacological protocol).

The administration of the humanized B7-1 antibody, humanized B7-2antibody and/or other drugs can occur simultaneously or sequentially intime. These compounds or compositions can be administered before, afteror at the same time. Thus, the term “co-administration” is used hereinto mean that the humanized B7-1 and/or B7-2 antibodies and/or othercompositions are administered at times to treat the diseases describedherein or induce tolerization (e.g., methotrexate, rapamycin,cyclosporin, steroids, anti-CD40 antibodies, and analogs thereof). Themethods of the present invention are not limited to the sequence inwhich the antibodies or compositions are administered, so long as theyare administered close enough in time to produce the desired effect.

The invention also pertains to methods for determining the presence,absence or level of B7-2 or B7-1 using a humanized anti-B7-2 or B7-1antibody, respectively. The presence or absence of B7-2 or B7-1 can bedetected in an assay (e.g., ELISA, radioimmunoassay (RIA) or FACSImmunohistochemistry). The assay can be a direct detection or anindirect detection (e.g. a competitive assay).

For example, to determine the presence or absence of B7-2 or B7-1 usingan ELISA assay in a suitable sample, the method comprises combining asuitable sample with a composition comprising a humanized or murineanti-B7-2 or B7-1 antibody as detector (e.g., biotinylated anti-B7-2 orB7-1 mAb and HRP-streptavidin, or HRP-conjugated anti-B7-2 or B7-1 mAb)and a solid support (e.g., a microtiter plate), having an anti-B7-2 orB7-1 capture antibody bound (directly or indirectly) thereto. Thedetector antibody can bind to a different B7-2 or B7-1 epitope from thatrecognized by the capture antibody, under conditions suitable for theformation of a complex between the anti-B7-2 or B7-1 antibodies and B7-2or B7-1, respectively. The method further comprises determining theformation of complex in the sample.

The presence of B7-2 or B7-1 can also be determined in aradioimmunoassay or a fluorescent assay. For example, the presence ofB7-2 or B7-1 can be assessed by an immunobinding assay comprisingobtaining a sample, contacting the sample with a composition comprisingan anti-B7-2 or B7-1 antibody (e.g., a humanized or murine anti-B7-2 orB7-1 antibody comprising a radioactive or fluorescent label; or ahumanized or murine anti-B7-2 or B7-1 antibody comprising a binding sitefor a second antibody which comprises a radioactive or fluorescentlabel), preferably in an amount in excess of that required to bind theB7-2 or B7-1, under conditions suitable for the formation of labeledcomplexes. The method further comprises determining (detecting ormeasuring) the formation of complex in the samples. Similarly, thepresent, absence or level of B7-1 or B7-2 can be determined usingFluorescent-Activated Cell Sorting (FACS) analysis, or histo-chemicalanalysis of tissues, using the humanized anti-B7-1 or B7-2 antibody ofthe present invention.

EXEMPLIFICATION

The present invention will now be illustrated by the following Examples,which are not intended to be limiting in any way.

Example 1 Cloning and Sequencing of Mouse 3D1 Variable Region cDNAs:

Mouse 3D1 (also referred to as HF2.3D1) heavy and light chain variableregion cDNAs were cloned from mRNA isolated from hybridoma cells usinganchored PCR (Co et al., J. Immunol. 148: 1149 (1992)). The 5′ primersused annealed to the poly-dG tails added to the cDNA, and the 3′ primersannealed to the constant regions. The amplified gene fragments were theninserted into the plasmid pUC18. Nucleotide sequences were determinedfrom several independent clones for both V_(L) and V_(H) cDNA. For theheavy chain, a single, unique sequence was identified, typical of amouse heavy chain variable region. For the light chain, two uniquesequences, both homologous to murine light chain variable regionsequences, were identified. However, one sequence was not functionalbecause of a missing nucleotide that caused a frame shift at the V-Jjunction, and was identified as the non-productive allele. The othersequence was typical of a functional mouse kappa chain variable region.The variable region cDNA sequences of the heavy chain and the functionallight chain and the translated amino acid sequences are shown in FIGS.1A-1B. The mouse V_(L) sequence belongs to Kabat's mouse kappa chainsubgroup I. The mouse V_(H) belongs to Kabat's heavy chain subgroupII(A).

Example 2 Design of Humanized 3D1 Variable Regions:

To retain the binding affinity of the mouse antibody in the humanizedantibody, the general procedures of Queen et al. were followed (Queen etal., Proc. Natl. Acad. Sci. USA 86: 10029 (1989), U.S. Pat. Nos.5,585,089 and 5,693,762, the teachings of which are incorporated hereinin their entirety). The choice of framework residues can be critical inretaining high binding affinity. In principle, a framework sequence fromany human antibody can serve as the template for CDR grafting; however,it has been demonstrated that straight CDR replacement into such aframework can lead to significant loss of binding affinity to theantigen (Tempest et al., Biotechnology 9: 266 (1992); Shalaby et al., J.Exp. Med. 17: 217 (1992)). The more homologous a human antibody is tothe original murine antibody, the less likely the human framework willintroduce distortions into the mouse CDRs that could reduce affinity.Based on a sequence homology, III-2R was selected to provide theframework for the humanized 3D1 heavy chain and H₂F for the humanized3D1 light chain variable region. Manheimer-Lory, A. et al., J. Exp. Med.174(6):1639-52 (1991). Other highly homologous human antibody chainswould also be suitable to provide the humanized antibody framework,especially kappa light chains from human subgroup 4 and heavy chainsfrom human subgroup 1 as defined by Kabat.

Normally the heavy chain and light chain from the same human antibodyare chosen to provide the framework sequences, so as to reduce thepossibility of incompatibility in the assembling of the two chains. TheII-I2R antibody shows a high homology to the 3D1 heavy and light chainsand thus, was chosen to provide the framework for the initial humanizedantibody model. The 3D1 light chain variable region, however, shows asignificantly higher homology to the H₂F framework compared to anyothers, including III-2R. Therefore, H₂F was chosen instead to providethe framework for the humanized 3D1 light chain variable region, whileIII-2R was selected to provide the framework for the heavy chainvariable region.

The computer programs ABMOD and ENCODE (Levitt et al., J. Mol. Biol.168: 595 (1983)) were used to construct a molecular model of the 3D1variable domain, which was used to locate the amino acids in the 3D1framework that are close enough to the CDRs to potentially interact withthem. To design the humanized 3D1 heavy and light chain variableregions, the CDRs from the mouse 3D1 heavy chain were grafted into theframework regions of the human III-2R heavy chain and the CDRs from themouse 3D1 light chain grafted into the framework regions of the humanH₂F light chain. At framework positions where the computer modelsuggested significant contact with the CDRs, the amino acids from themouse antibody were substituted for the original human framework aminoacids. For humanized 3D1, this was done at residues 27,30, 48, 67, 68,70 and 72 of the heavy chain and at residue 22 of the light chain.Furthermore, framework residues that occurred only rarely at theirpositions in the database of human antibodies were replaced by a humanconsensus amino acid at those positions. For humanized 3D1 this was doneat residue 113 of the heavy chain and at residue 3 of the light chain.

The sequence of the humanized 3D1 antibody heavy chain and light chainvariable regions is shown in FIGS. 2A-2B. However, many of the potentialCDR-contact residues are amenable to substitutions of other amino acidsthat may still allow the antibody to retain substantial affinity to theantigen. Table 1 lists a number of positions in the framework wherealternative amino acids may be suitable (LC=light chain, HC=heavychain). The position specified in the table is the number of amino acidsfrom the first amino acid of the mature chain, which is indicated by adouble underline (FIGS. 2A-2B). For example, position LC-22 is thetwenty second amino acid beginning from the doubled underlined AsparticAcid, D, (or the forty second amino acid from the start codon).

TABLE 1 Amino Acids Substitutes and/or Alternatives Position Humanized3D1 Alternatives LC-22 S N HC-27 Y G HC-30 T S HC-48 I M HC-67 K R HC-68A V HC-70 M I HC-72 V A

Likewise, many of the framework residues not in contact with the CDRs inthe humanized 3D1 heavy and light chains can accommodate substitutionsof amino acids from the corresponding positions of III-2and H2Fframeworks, from other human antibodies, from the mouse 3D1 antibody, orfrom other mouse antibodies, without significant loss of the affinity ornon-immunogenicity of the humanized antibody. Table 2 lists a number ofadditional positions in the framework where alternative amino acids maybe suitable.

TABLE 2 Framework Region Amino Acid Substitutes and/or AlternativesPosition Humanized 3D1 Alternatives LC-3 V Q HC-113 T I

Selection of various alternative amino acids may be used to produceversions of humanized 3D1 that have varying combinations of affinity,specificity, non-immunogenicity, ease of manufacture, and otherdesirable properties. Thus, the examples in the above tables are offeredby way of illustration, not of limitation.

Example 3 Construction of Humanized 3D1

Once the humanized variable region amino acid sequences had beendesigned, as described above, genes were constructed to encode them,including signal peptides, splice donor signals and appropriaterestriction sites (FIG. 2A-2B). The light and heavy chain variableregion genes were constructed and amplified using eight overlappingsynthetic oligonucleotides ranging in length from approximately 65 to 80bases (see He et al., J. Immunol. 160: 1029 (1998)). The oligos wereannealed pairwise and extended with the Klenow fragment of DNApolymerase I, yielding four double-stranded fragments. The resultingfragments were denatured, annealed, and extended with Klenow, yieldingtwo fragments. These fragments were denatured, annealed pairwise, andextended once again, yielding a full-length gene. The resulting productwas amplified by polymerase chain reaction (PCR) using Taq polymerase,gel-purified, digested with XbaI, gel-purified again, and subcloned intothe XbaI site of the pVk for the expression of light chain and pVg4 orpVg2.M3 for the expression of heavy chains. The pVk vector for kappalight chain expression has been previously described (See Co et al., J.Immunol. 148:1149 (1992)). The pVg4 vector for the γ4 heavy chainexpression was constructed by replacing the XbaI-BamHI fragment of pVg1containing the γ1 constant region gene (See Co et al., J. Immunol. 148:1149 (1992)) with an approximately 2000 bp fragment of the human g4constant region gene (Ellison and Hood, Proc. Natl. Acad. Sci. USA 79:1984 (1982)) that extended from the HindIII site preceding the C_(H)1exon of the γ4 gene to 270 bp after the NsiI site following the C_(H)4exon of the gene. The pVg2.M3 vector for the γ2 heavy chain expressionwas described in Cole, et al., J. Immunol. 159: 3613 (1997). The pVg2.M3is mutated from the human wildtype IgG2 by replacing the amino acids Valand Gly at positions-234 and 237 with Ala. This variant has a reducedinteraction with its Fc receptors and thus has minimal antibody effectoractivity.

The structure of the final plasmids was verified by nucleotidesequencing and restriction mapping. All DNA manipulations were performedby standard methods well-known to those skilled in the art.

Two humanized 3D1, an IgG4 and an IgG2.M3, were generated forcomparative studies. To construct a cell line producing humanized 3D1(IgG4 or IgG2.M3), a light chain and the respective heavy chain plasmidswere transfected into the mouse myeloma cell line Sp2/0-Ag14 (A.T.C.C.Accession No. CRL 1581). Plasmids were also transfected into CHO cellsusing known methods in the art. Before transfection, the heavy and lightchain-containing plasmids were linearized using restrictionendonucleases. The kappa chain and the γ2 chain were linearized usingFspI; the γ4 chain was linearized using BstZ17I. Approximately 20 μg ofthe light chain and a heavy chain plasmid was transfected into 1×10⁷cells in PBS. Transfection was by electroporation using a Gene Pulserapparatus (BioRad) at 360 V and 25 μFD capacitance according to themanufacturer's instructions. The cells from each transfection wereplated in four 96-well tissue culture plates, and after two days,selection medium (DMEM, 10% FCS, 1×HT supplement (Sigma), 0.25 mg/mLxanthine, 1 μg/mL mycophenolic acid) was applied.

After approximately two weeks, the clones that appeared were screenedfor antibody production by ELISA. Antibody from a high-producing clonewas prepared by growing the cells to confluency in regular medium (DMEMwith 10% FCS), then replacing the medium with a serum-free medium(Hybridoma SMF; Gibco) and culturing until maximum antibody titers wereachieved in the culture. The culture supernatant was run through aprotein A-Sepharose column (Pharmacia); antibody was eluted with 0.1 MGlycine, 100 mM NaCl, pH 3, neutralized and subsequently exchanged intophosphate-buffered saline (PBS). The purity of the antibody was verifiedby analyzing it on an acrylamide gel, and its concentration wasdetermined by an OD₂₈₀ reading, assuming 1.0 mg of antibody protein hasan OD₂₈₀ reading of 1.4.

Example 4 Affinity of Humanized Anti-B7-2 Antibody

Competitive Binding Assay:

The relative affinities, of the murine and humanized 3D1 antibodies forthe B7-2 antigen were determined by competitive binding assays.Three-fold serial dilutions of unlabeled humanized or murine 3D1antibodies were mixed with a fixed amount of radio-iodinated murine 3D1antibody (40,000-50,000 cpm per test in PBS containing 2% fetal calfserum).

1×10⁵ CHO cells expressing cell surface rhB7-2 (CHO/hB7-2) were addedsubsequently and the mixture (in a total volume of 200 μL) was incubatedfor 2 hr at 4° C. with gentle shaking. The cell-antibody suspension wasthen transferred to Sarstedt Micro Tubes (part #72.702) containing 100μL of 80% dibutyl phthalate-20% olive oil. After centrifugation in amicrofuge, the Sarstedt tubes were plunged into dry ice for severalminutes. Cell-bound ¹²⁵I was determined by clipping tips of each tube(containing cell pellets) into scintillation vials and counting in agamma counter. Bound and free counts were determined and the ratioplotted against the concentrations of the cold competitor antibodiesaccording to the method of Berzofsky and Berkower (J. A. Berzofsky andI. J. Berkower, in Fundamental Immunology 9ed. W. E. Paul), Raven Press(New York), 595 (1984)).

Cell Line:

Recombinant Chinese Hamster Ovary (CHO) cell lines expressing hB7-2 ontheir membrane surfaces were cloned from cells transfected with B7-2cDNA sequence and G418 resistance. Expression of hB7-2 on the CHO cellsurface over many passages under selective pressure has been confirmedusing murine anti-B7 antibodies and FACS analysis.

Preparation of ¹²⁵I Labeled Anti-hB7 mAb and Characterization:

Anti-hB7 antibodies were labeled with ¹²⁵I by reaction with¹²⁵I-Bolton-Hunter reagent according to manufacturers instructions(Amersham Corp., Arlington Hts, Ill.). Protein was separated from freereagent with a NAP-25 column. An HPLC size-exclusion column was used toconfirm that the antibodies remained intact and were not aggregated, andto measure protein concentration against standards prepared fromnon-labeled antibody. Labeling typically resulted in 4 to 8 microcuriesper microgram of protein, or approximately 30 to 60% of the antibodymolecules labeled.

Results:

The competitive binding graph is shown in FIG. 3. Each data pointrepresents the average of triplicate determinations. Results showed thatboth humanized IgG4 and humanized IgG2.M3 anti-human B7-2 antibodieshave a similar high binding affinity as the murine anti-human B7-2antibody (approximately 1×10⁹ M⁻¹), indicating no loss of affinity forB7-2 in the humanization of 3D1. Both murine and humanized anti-B7-2recognize cell surface expressed hB7-2 with high affinity.

Example 5 Direct Binding of Humanized Anti-B7 mAbs to CHO/hB7 Cells

Cell Binding Assay:

Binding assays were begun by plating cells onto 96-well tissue cultureplates at 10,000 CHO/hB7-2 cells per well. Two days later, adherentcells were gently washed with assay buffer containing nonfat dry milkprotein (for blocking nonspecific binding) and sodium azide (to preventinternalization of antibodies by cells). For direct binding assays,¹²⁵I-labeled anti-B7 antibodies (¹²⁵I-murine anti-human B7-2; 826cpm/fmol; humanized anti-human B7-2, 883 cpm/fmol) were serially dilutedin assay buffer and incubated on cells overnight, allowing antibodies tobind to cell-surface B7 and come to equilibrium. Unbound antibody wasgently washed from cells, and bound ¹²⁵I-labeled antibody was detectedusing an ¹²⁵I scintillant and photodetector system. Non-specific bindingto CHO cells was determined for each dilution in the same manner, but oncells expressing the B7-1 molecule that is not recognized by theantibody being tested.

Results:

The direct binding graph is shown in FIG. 4. The data, means oftriplicate wells with nonspecific binding subtracted, were fit to ahyperbolic saturation curve using Graphpad PrismJ software. K_(D) of theantibodies determined as the concentration corresponding to half-maximalbinding indicated that the murine and humanized anti-B7-2 mAbs hadsimilar and high binding affinities (˜10⁻⁹ m) for B7-2. Both murine andhumanized anti-B7-2 antibodies recognize cell surface expressed hB7-2with high affinity.

Example 6 Binding of Humanized Anti-B7 mAbs to Protein Ligands

Affinity Determination by BIACORE®:

The BIACORE® biosensor (BIACORE®; Uppsalla, Sweden) was used todetermine binding kinetics of murine and humanized anti-B7-2 humanantibodies to human B7-2Ig. Hunan B7-2Ig (hB7-2Ig) was immobilized ontothe dextran matrix of a BIACORE® sensor chip. Humanized and murineanti-human B7-2 were tested at 200, 100, 50, and 20 nM. Each dilutionwas tested 4 times per run and a total of three separate runs performed.Anti-human B7-2 antibody binding was measured in real time by SurfacePlasmon Resonance (SPR) and global analysis was performed using thebivalent binding model in BIA evaluation software (version 3.1). Foreach sample, the association (k_(a)), dissociation (k_(d)), andequilibrium dissociation constant (K_(D)) were determined.

Preparation of hB7-2 Ig:

A soluble form of hB7-2Ig was recovered from culture medium of CHO cellsengineered to secrete this protein. Recombinant hB7-2Ig was derived byfusing the DNA coding sequences corresponding to the extracellulardomain of B7-2 gene to the hinge-CH2—CH3 domains of the human IgG1 heavychain. Recombinant hB7-2Ig was purified from the culture medium byprotein A.

Results:

Table 3 reports the mean values obtained for both murine and humanizedanti-human B7-2 mAbs. The binding constants for the murine and humanizedanti-B7-2 mAbs determined by SPR shows that the murine and humanizedforms of the anti-B7-2 mAbs are similar and that the murine anti-B7-2mAb has a slightly higher binding constant for the immobilized hB7-2 Igthan does the humanized anti-B7-2. The approximately 2.8 fold higheraffinity calculated for the murine anti-B7-2 mAb may represent a real,but slight difference between the murine and humanized anti-B7-2 mAbsintroduced during the humanization process. Another possibility may bedue to technical variation in the preparation, processing and analysisof these antibodies. As shown in Examples 4, 5, and 7, a difference wasnot observed in humanized hB7-2 binding affinity in cell based assays.

TABLE 3 Affinity of anti-B7-2 mAbs as determined by BIACORE ® mAb MeanK_(D) murine Anti-B7-2 1.8 × 10⁻⁹ M humanized 5.1 × 10⁻⁹ M Anti-B7-2

Example 7 Inhibition of T Cell Costimulation by Humanized Anti-B7-2

CD28⁺T cell/CHO-B7 Proliferation Assay

CD28⁺T cells, isolated as described herein, were washed once andresuspended in RPMI complete medium, supplemented with 2 ng/mL PMA(Calbiochem), to a cell density of 5×10⁵ cells/mL. The CD28⁺T cells (100μL, 5×10⁴ cells) were added to the antibody/CHO/hB7-2 mixture (seebelow), incubated for 3 days at 37° C., 5% CO₂, and T cell proliferationwas measured by pulsing for the last 6 hours of culture with 1 uCi of[³H]-thymidine (NEN, Boston, Mass.). The cells were harvested on afilter and the incorporated radioactivity was measured in ascintillation counter.

Materials:

CD28′ T cells were isolated by negative selection with immunoabsorptionfrom human peripheral blood lymphocytes, as described (June et al., Mol.Cell. Biol. 7:4472-4481 (1987)). Buffy coats were obtained byleukophoresis of healthy human donors and peripheral blood lymphocytes(PBL) were isolated by density gradient centrifugation. Monocytes weredepleted from the PBL by plastic absorption. CD28⁺T cells were isolatedfrom the non-adherent cells by negative selection using antibodies toCD11, CD20, CD16 and CD 14, (this set of antibodies will coat all Bcells, monocytes, large granular lymphocytes, and CD28⁻ T cells) andmagnetic bead separation using goat anti-mouse immunoglobulin-coatedmagnetic particles.

CHO/hB7-2 cells were detached from the tissue culture plates byincubation in phosphate-buffered saline without Ca²⁺ and Mg²⁺ (PBS) with0.5 mM EDTA, washed, and fixed with freshly prepared paraformaldehyde.

Various concentrations of anti-B7-2 antibody (in duplicate) werepreincubated for 1 hour at 37° C., 5% CO₂ with 1×10⁴ CHO/hB7-2 cells in100 μL RPMI complete medium (RPMI 1640 medium, 10% fetal bovine serum(FBS), 100 U/mL penicillin, 100 μg/mL streptomycin) in a microtiterplate (flat-bottom, 96-well, Costar, Cambridge, Mass.).

Results:

FIG. 5 shows the results of the inhibition of human CD28⁺T cellproliferation by the murine and humanized anti-hB7-2 mAbs. Bothantibodies exhibit dose dependent inhibition of B7-2 driven T cellproliferation with similar IC₅₀ (Inhibitory concentration 50%; amount ofantibody required to inhibit the maximal T cell proliferation by 50%)values of 72 pm (murine anti-hB7-2) and 50 pm (humanized anti-hB7-2)indicating that both antibodies were similar and very effective ininhibiting the B7-2 T cell stimulatory signal. This demonstrates thatthe high affinity anti-B7-2 mAbs can block B7-2 functionality byinhibiting (e.g., preventing) the activation and/or proliferation ofhuman T cells. These mAbs are expected to exhibit similar capability inin vivo use to inhibit T cell response.

Example 8 Cloning and Sequencing of Mouse 1F1 Variable Region cDNAs

Mouse 1F1 heavy and light chain variable region cDNAs were cloned frommRNA isolated from hybridoma cells using anchored PCR (Co et al., J.Immunol. 148: 1149 (1992)). The 5′ primers used annealed to the poly-dGtails added to the cDNA, and the 3′ primers annealed to the constantregions. The amplified gene fragments were then inserted into theplasmid pUC19. Nucleotide sequences were determined from severalindependent clones for both V_(H) and V_(L) cDNA. For the heavy chain, asingle, unique sequence was identified, typical of a mouse heavy chainvariable region. For the light chain, two unique sequences, bothhomologous to mouse light chain variable regions, were identified.However, one sequence was not functional because of a missing nucleotidethat caused a frame shift at the V-J junction, and was identified as thenon-productive allele. The other sequence was typical of a functionalmouse kappa chain variable region. The variable region cDNA sequences ofthe heavy chain and the functional light chain, and the translated aminoacid sequences are shown in FIGS. 6A and 6B, respectively. The mouseV_(H) belongs to Kabat's heavy chain subgroup II(C). The mouse V_(K)sequence belongs to Kabat's mouse kappa chain subgroup IV.

Example 9 Design of Humanized 1F1 Variable Regions

To retain the binding affinity of the mouse antibody in the humanizedantibody, the general procedures of Queen et al. were followed (Queen etal., Proc. Natl. Acad. Sci. USA 86: 10029 (1989) and U.S. Pat. Nos.5,585,089 and 5,693,762). The choice of framework residues can becritical in retaining high binding affinity. In principle, a frameworksequence from any human antibody can serve as the template for CDRgrafting; however, it has been demonstrated that straight CDRreplacement into such a framework can lead to significant loss ofbinding affinity to the antigen (Tempest et al., Biotechnology 9: 266(1992); Shalaby et al., J. Exp. Med. 17: 217 (1992)). The morehomologous a human antibody is to the original mouse antibody, the lesslikely that the human framework will introduce distortions into themouse CDRs that could reduce affinity. Based on a sequence homologysearch against the Kabat antibody sequence database (Kabat et al.,Sequences of Proteins of Immunological Interest, 5^(th) ed., U.S.Department of Health and Human Services (1991)), III-2R (Manheimer-Loryet al., J. Exp. Med. 1276: 309 (1992)) and the original reference,Manheier-Lory et al. J Exp. Med. 174: 1639-1652(1991), cited therein)was selected to provide the framework for both the humanized 1F1 heavychain variable region and for the humanized 1F1 light chain variableregion. Other highly homologous human antibody chains would also besuitable to provide the humanized antibody framework, especially heavychains from human subgroup 1 and kappa light chains from human subgroup1 as defined by Kabat.

Normally the heavy chain and light chain from the same human antibodyare chosen to provide the framework sequences, so as to reduce thepossibility of incompatibility in the assembly of the two chains. TheIII-2R antibody shows a high homology to the 1F1 heavy and light chainsand thus was chosen to provide the framework for the humanized antibody.The humanized 1F1 heavy chain variable domain has 69 residues out of 87framework residues that are identical to those of the mouse 1F1 heavychain framework, or 79% sequence identity. The humanized 1F1 light chainvariable domain has 55 residues out of 80 framework residues that areidentical to those of the mouse 1F1 light chain framework, or 69%sequence identity.

The computer programs ABMOD and ENCAD (Levitt et al., J. Mol. Biol. 168:595 (1983)) were used to construct a molecular model of the 1F1 variabledomain, which was used to locate the amino acids in the 1F1 frameworkthat are close enough to the CDRs to potentially interact with them. Todesign the humanized 1F1 heavy and light chain variable regions, theCDRs from the mouse 1F1 heavy chain were grafted into the frameworkregions of the human III-2R heavy chain and the CDRs from the mouse 1F1light chain were grafted into the framework regions of the human III-2Rlight chain. At framework positions where the computer model suggestedsignificant contact with the CDRs, the amino acids from the mouseantibody were substituted for the original human framework amino acids.For humanized 1F1, this was done at residues 1, 24, 27, 28, 29, 30, 48,67, and 68 of the heavy chain and at residues 47 and 72 of the lightchain. Furthermore, framework residues that occurred only rarely attheir positions in the database of human antibodies were replaced byhuman consensus amino acids at those positions. For humanized 1F1 thiswas done at residues 16, 74, and 113 of the heavy chain and at residue44 of the light chain. Overall, the humanized 1F1 heavy chain variabledomain has 88 residues that are identical to the human III-2R heavychain variable domain, and the humanized 1F1 light chain variable domainhas 88 residues that are identical to the III-2R light chain variabledomain.

The sequences of the humanized 1F1 antibody heavy chain and light chainvariable regions are shown in FIGS. 7A and 7B. However, many of thepotential CDR-contact residues are amenable to substitutions of otheramino acids that still allow the antibody to retain substantial affinityto the antigen. Table 4 lists a number of positions in the frameworkwhere alternative amino acids may be suitable (LC=light chain, HC=heavychain).

TABLE 4 Position Humanized 1F1 Alternatives HC-1 E Q HC-24 P A HC-27 FG, Y HC-28 N T HC-29 I F HC-30 K S, T HC-48 I M HC-67 K R HC-68 A VLC-72 Y F

Likewise, many of the framework residues not in contact with the CDRs inthe humanized 1F1 heavy and light chains can accommodate substitutionsof amino acids from the corresponding positions of the III-2R framework,from other human antibodies, from the mouse 1F1 antibody, or from othermouse antibodies, without significant loss of the affinity ornon-immunogenicity of the humanized antibody. Table 5 lists a number ofadditional positions in the framework where alternative amino acids maybe suitable.

TABLE 5 Position Humanized 1F1 Alternatives HC-16 A S HC-74 T K HC-113 TI LC-44 A S, V

Selection of various alternative amino acids may be used to produceversions of humanized 1F1 that have varying combinations of affinity,specificity, non-immunogenicity, ease of manufacture, and otherdesirable properties. Thus, the examples in the above tables are offeredby way of illustration, not of limitation.

Example 10 Construction of Humanized 1F1

Once the humanized variable region amino acid sequences had beendesigned as described above, genes were constructed to encode them,including signal peptides, splice donor signals and appropriaterestriction sites (FIGS. 7A and 7B). The heavy and light chain variableregion genes were constructed and amplified using eight overlappingsynthetic oligonucleotides ranging in length from approximately 65 to 80bases (see He et al., J. Immunol. 160: 1029 (1998)). The oligos wereannealed pairwise and extended with the Klenow fragment of DNApolymerase I, yielding four double-stranded fragments. The resultingfragments were denatured, annealed pairwise, and extended with Klenow,yielding two fragments. These fragments were denatured, annealedpairwise, and extended once again, yielding a full-length gene. Theresulting product was amplified by polymerase chain reaction (PCR) usingTaq polymerase, gel-purified, digested with XbaI, gel-purified again,and subcloned into the XbaI site of pVg2.M3 for the expression of heavychain, and pVk for the expression of light chain. The pVg2.M3 vector forhuman γ 2 heavy chain expression has been previously described (Cole etal., J. Immunol. 159: 3613 (1997)). The human γ2 constant region in thepVg2.M3 plasmid is mutated from the wildtype human γ2 constant region byreplacing the amino acids Val and Gly at positions 234 and 237 with Ala.This variant has a reduced interaction with its Fc receptors and thushas minimal antibody effector activity. The pVk vector for human kappalight chain expression has been previously described (see Co et al., J.Immunol. 148: 1149 (1992)).

The structures of the final plasmids were verified by nucleotidesequencing and restriction mapping. All DNA manipulations were performedby standard methods well-known to those skilled in the art.

An IgG2.M3 form of the humanized 1F1 antibody was generated for bindingstudies. To construct a cell line producing humanized 1F1, the heavy andlight chain plasmids were transfected into mouse myeloma cell lineSp2/0-Ag14 (A.T.C.C. Accession No. CRL 1581). Before transfection, theheavy and light chain plasmids were linearized using restrictionendonucleases. The γ2 heavy chain plasmid and the kappa light chainplasmid were linearized using FspI. Approximately 40 μg of the heavychain plasmid and 20 μg of the light chain plasmid were transfected into1×10⁷ cells in PBS. Transfection was by electroporation using a GenePulser apparatus (BioRad) at 360 V and 25 μFD capacitance according tothe manufacturer's instructions. The cells from each transfection wereplated in four 96-well tissue culture plates, and after two daysselection medium (DMEM, 10% FCS, 1×HT supplement (Sigma), 0.25 mg/mLxanthine, 1 μg/mL mycophenolic acid) was applied.

After approximately two weeks, the clones that appeared were screenedfor antibody production by ELISA. Antibody from a high-producing clonewas prepared by growing the cells to confluency in regular medium (DMEMwith 10% FCS), then replacing the medium with a serum-free medium(Hybridoma SFM; GIBCO) and culturing until maximum antibody titers wereachieved in the culture. The culture supernatant was run through aprotein A-Sepharose column (Pharmacia); antibody was eluted with 0.1 MGlycine, 100 mM NaCl, pH 3, neutralized, and subsequently exchanged intophosphate-buffered saline (PBS). The purity of the antibody was verifiedby analyzing it on an acrylamide gel, and its concentration wasdetermined by an OD₂₈₀ reading, assuming 1.0 mg of antibody protein hasan OD₂₈₀ reading of 1.4.

An IgG4 form of the humanized 1F1 antibody was also generated andpurified following the methods described above.

In order to permit high level expression in CHO cells, the completehumanized human 1F1 (h1F1) and human 3D1 (h3D1) light chain and heavychain genes were each independently subcloned into the selectable,amplifiable expression vector pED (Kaufman R. J., et al., Nucl AcidsRes., 19:4485-4490 (1991)). The pED-derived expression plasmids weresequenced to confirm that they encoded the appropriate h1F1 and h3D1light and heavy chains. The penultimate amino acid of the IgG2 m3 CH3domain was found to be serine in contrast to the glycine residuereported at this position for all published IgG2 and IgG1 sequences.This serine was replaced with the more common glycine for the pEDexpression constructs. The h1F1 light chain and heavy chain expressionplasmids (pED.1F1v2KA and pED.1F1v2G2 m3gly) were linearized andcotransfected into the CHO PA-DUKX.153.8 cell line, which has beenpre-adapted for growth in serum-free suspension culture (Sinacore M. S.et al., Biotechnol. Bioeng. 52:518-528 (1996)). Cell lines expressingh3D1 were generated in the same manner by cotransfection with linearizedplasmids pED.3D1KA and pED.3D1G2 m3gly. In each case, stable integrationof light chain and heavy chain genes into the CHO cell genome, followedby methotrexate selection and amplification, resulted in recombinanth1F1 or h3D1 cell lines. The CHO cell lines expressing anti-B7-1 oranti-B7-2 were cultured in serum-free growth medium and the secretedantibodies purified from the conditioned culture supernatant bychromatography on protein A sepharose. Bound antibody was eluted in acidbuffer followed by neutralization to pH 7.0 with. The purified antibodywas buffer exchanged into PBS and sterile filtered.

Example 11 Properties of Humanized 1F1

The affinity of the mouse and humanized 1F1 antibodies for the B7-1antigen was determined by competitive binding with radio-iodinatedhumanized 1F1 antibody. Three-fold serial dilutions of unlabeled mouseor humanized 1F1 antibodies were mixed with a fixed amount ofradio-iodinated humanized 1F1 antibody (40,000-50,000 cpm per test) inPBS containing 2% fetal calf serum, 0.1% sodium azide. 3×10⁴ CHO cellsexpressing cell surface rhB7-1 were added subsequently and the mixture(in a total volume of 200 μL) was incubated for 2 hr at 4° C. withgentle shaking. The cell-antibody suspension was then transferred toSarstedt Micro Tubes (part #72.702) containing 100 μL of 80% dibutylphthalate-20% olive oil. After centrifugation in a microfuge, theSarstedt tubes were plunged into dry ice for several minutes. Cell-bound¹²⁵I was determined by clipping the tips of each tube (containing cellpellets) into scintillation vials and counting in a gamma counter. Boundand free counts were determined and the ratio plotted against theconcentrations of the cold competitor antibodies according to the methodof Berzofsky and Berkower (J. A. Berzofsky and I. J. Berkower, inFundamental Immunology, W. E. Paul, Raven Press, New York, pp. 595-644(1984)).

The competitive binding graphs are shown in FIG. 8. Each data pointrepresents the average of triplicate determinations. The results showedthat the IgG2.M3 antibody has a similar binding affinity to that of themouse antibody (approximately 1×10⁹ M⁻¹), indicating no loss of affinityin the humanization of 1F1.

The affinity of the mouse and humanized 1F 1 antibodies for the B7-1antigen was confirmed by Scatchard analysis of binding of radiolabeledantibodies. Two-fold serial dilutions of radiolabeled mouse or humanized1F1 antibodies were incubated with 5×10⁴ CHO cells expressing cellsurface rhB7-1 in PBS containing 2% FCS, 0.1% sodium azide in a totalvolume of 200 μL. The mixture was incubated for 6 hr at 4° C. withgentle shaking. The cell-antibody suspension was then transferred toSarstedt Micro Tubes (part #72.702) containing 100 μL of 80% dibutylphthalate-20% olive oil. After centrifugation in a microfuge, theSarstedt tubes were plunged into dry ice for several minutes. Cell-bound¹²⁵I was determined by clipping the tips of each tube (containing cellpellets) into scintillation vials and counting in a gamma counter. Boundand free counts were determined and the ratio plotted against theconcentration of bound antibody following the method of Scatchard(Scatchard, Ann. N.Y. Acad. Sci. 51: 660 (1949)). A least squares methodwas used to fit a line to the data, and the apparent Ka was determinedfrom the slope of the line.

The Scatchard plots are shown in FIGS. 9A and 9B. Each data pointrepresents the average of duplicate determinations. The results showedthat the IgG2.M3 antibody has a similar binding affinity to that of themouse antibody (approximately 1×10⁹ M-1), confirming no loss of affinityin the humanization of 1F1.

Example 12 Affinity of Humanized Anti-B7-1 Monoclonal Antibody

Competitive Binding Assay:

The relative affinities of the murine and humanized anti-B7-1 (1F1)antibodies for the B7-1 antigen were determined by competitive bindingassays. Three-fold serial dilutions of the unlabeled murine or humanizedanti-B7-1 mAbs were mixed with a fixed amount of radio-labeled murineanti-B7-1 mAb (¹²⁵I-anti-B7-1, 2800 cpm/fmol; 40,000-50,000 cpm/test inPBS containing 2% fetal calf serum). 1×10⁵ CHO/hB7-1 cells expressinghB7-1 on their surface were added subsequently and the mixture (totalvolume=200 μL) incubated for 2 hrs at 4° C. with gentle shaking. Thecell antibody mixture was then transferred to Sarstedt Micro tubes (Part# 72.702) containing 100 μL of 80% dibutyl phthlate-20% olive oil. Aftercentrifugation in a microfuge, the Sarstedt tubes were plunged into dryice for several minutes. Cell bound ¹²⁵I-labeled mAb was determined byclipping the cell pellet containing tips of each tube into scintillationvials and counting in a gamma counter. Bound and free counts weredetermined. The bound counts were plotted against the concentration ofthe cold competitor mAbs.

CHO/hB7-1 Cell Line:

A recombinant Chinese Hamster Ovary (CHO) cell line expressing hB7-1 onits surface was cloned form CHO cells transfected with the hB7-1 cDNAsequence and G418 resistance marker. Stable expression of the hB7-1 onthe CHO cell surface over many passages under selective pressure hasbeen confirmed using the murine anti-B7-1 mAb and FACS analysis.

Preparation of ¹²⁵I-Labeled Anti-B7-1 mAbs:

Murine and Humanized anti-B7-1 mAbs were labeled with ¹²⁵Iodine byreaction with ¹²⁵I-Bolton Hunter reagent according to the manufacturers'instructions (Amersham Corp, Arlington Heights, Ill.). Protein wasseparated from free reagent with a NAP-25 column. HPLC size-exclusionchromatography was used to verify antibody integrity and aggregationstate post labeling and to determine protein concentration. Labelingtypically resulted in 4 to 8 microcuries ¹²⁵I/μg of mAb with anestimated 30 to 60% of the antibody molecules labeled. The murine andhumanized anti-B7-1 mAbs had a specific activity of 2800 cpm/fmol and950 cpm/fmol, respectively.

Results:

The graphical representation of the competitive binding data is shown inFIG. 10. Each data point represents the average of triplicatedeterminations. Results show that both the humanized anti-B7-1 mAb andthe murine anti-B7-1 mAb from which it was derived have a similar highaffinity for B7-1 (approximately 1×10⁻⁹ M) indicating no loss ofaffinity for the humanized anti-B7-1 mAb. Both the murine and humanizedanti-B7-1 compete similarly and effectively with labeled murineanti-B7-1 mAb for binding to cell surface expressed B7-1.

Example 13 Direct Binding of Humanized Anti-B7-1 mAb to B7-1

Cell Binding Assay

Binding assays were begun by plating CHO/hB7-1 cells onto 96 well tissueculture plates at 10,000 cells/well in complete medium and the platesincubated at 37° C. for two days. Adherent cells were washed gently withassay buffer (PBS containing nonfat dry milk and sodium azide). Murineand humanized anti-B7-1 mAbs labeled with ¹²⁵I were serially diluted inassay buffer and incubated with the cells overnight at 4° C. to allowbiding to reach equilibrium. Unbound labeled antibody was removed fromthe cells by a series of gentle washings with assay buffer and the bound125I-labeled antibody was detected using a ¹²⁵I scintillant and detectorsystem. Non-specific binding was determined for each dilution of labeledantibody in an identical manner as described above except that thetarget CHO cells expressed hB7-2 which is not bound by either the murineor humanized anti-B7-1 mAbs.

Results:

The graphical depiction of the results of the direct binding experimentis shown in FIG. 11. The mean value of triplicate determinations minusthe non-specific binding was calculated and fit to a hyperbolicsaturation binding curve using Graphpad Prism software. Bindingconstants (K_(D)), determined as the concentration of antibodycorresponding to half-maximal saturation of binding sites, for both themurine and humanized antibodies indicated that both antibodies hadsimilar and high affinities for B7-1 (approximately 10⁻⁹M). Both murineand humanized anti-B7-1 mAbs recognize cell surface expressed humanB7-1.

Example 14 Binding of Murine and Humanized Anti-B7-1 mAbs to ProteinLigands

Affinity Determination by BIACORE®:

The BIACORE® biosensor (BIACORE®, Uppsalla, Sweden) was used todetermine binding kinetics of murine and humanized anti-B7-1 monoclonalantibodies to human B7-1Ig (hB7-1Ig) protein. Human B7-1Ig wasimmobilized onto the dextran matrix of a BIACORE® sensor chip. Humanizedand murine anti-B7-1 mAbs were tested at 200, 100, 50, and 20 nM on theimmobilized hB7-1Ig. Each mAb dilution was tested 4 times per run and atotal of three separate runs were performed. Anti-human B7-1Ig bindingwas measured in real time by Surface Plasmon Resonance (SPR) and globalanalysis was performed using the bivalent binding model in BIAevaluation software (Version 3.1). For each sample, the association(k_(a)) dissociation (k_(d)) and equilibrium dissociation constant(K_(D)) were determined.

Table 6 reports the mean values determined for both the murine andhumanized anti-B7-1 mAbs. The binding constants for the murine andhumanized anti-B7-mAbs determined by SPR shows that the murine andhumanized forms of the anti-B7-1 mAbs are similar and that the murineanti-B7-1 mAb had a slightly higher binding affinity for the hB7-1Igprotein than did the humanized anti-B7-1. The 5 fold higher bindingaffinity found for the murine anti-B7-1 mAb may represent a real butslight difference between the murine and humanized anti-B7-1 mAbsintroduced during the humanization process. Alternatively, technicalvariations in the preparation, processing, and analysis of theindividual mAbs may explain these minor differences. As shown inExamples 12, 14, and 19, no difference was observed between the murineand humanized anti-B7-1 mAbs in cell binding or functional assays.

TABLE 6 Affinity of anti-B7-1 mAbs as determined by BIACORE ® mAb MeanK_(D) Std deviation Murine anti-B7-1 5.6 × 10⁻¹⁰ M 1.9 × 10⁻¹⁰ MHumanized anti-B7-1 2.8 × 10⁻⁹ M 1.2 × 10⁻⁹ MPreparation of hB7-1Ig:

A soluble form of hB7-1Ig was recovered from culture medium of CHO cellsengineered to secrete this protein. Recombinant hB7-1Ig was derived byfusing the DNA sequences encoding the extracellular domain of hB7-1 geneto the hinge-CH2—CH3 domains of human IgG1 heavy chain. Recombinantprotein was purified from culture medium by protein A chromatography.

Example 15 Inhibition of T Cell Costimulation by Humanized B7-1 mAb

CD28⁺ T Cell/CHO-B7 Proliferation Assay

CD28⁺ T cells, isolated as described herein, were washed once andresuspended in RPMU complete medium supplemented with 2 ng/mL PMA(Calbiochem) to a cell density of 5×10⁴ cells/mL. The CD28⁺ T cells (100μL; 5×10⁴ cells) were added to the antibody/CHO/hB7-1 cell mixture (seebelow), incubated for 3 days at 37° C., 5% CO₂, and the T cellproliferation measured by pulsing for the last 6 hours of culture with 1uCi of [³H]-thymidine (NEN, Boston, Mass.). The cells were harvested ona filter and the incorporated radioactivity was measured byscintillation counting.

Materials:

CD28⁺ human T cells were isolated by negative selection withimmunoabsorption from human peripheral blood lymphocytes as described(June et al., Mol. Cell Biol. 7:4472-4481 (1987)). Briefly, buffy coatswere obtained by leukophoresis of healthy human donors and theperipheral blood lymphocytes (PBL) were isolated by density gradientcentrifugation. Monocytes were depleted from the PBLs by adsorption ontoplastic. CD28⁺ T cells were isolated from the non-adherent cells benegative selection using antibodies to CD11, CD20, CD16, and CD14 (thisset of antibodies will coat B cells, monocytes, large granularlymphocytes, and CD28⁻ T cells) and magnetic bead separation using goatanti-mouse immunoglobulin-coated magnetic beads.

CHO/hB7-1 cells were detached from the tissue culture plates byincubation with phosphate-buffered saline lacking Ca²⁺ or Mg²⁺ with 0.5mM EDTA. The cells were washed and then fixed with freshly preparedparaformaldehyde.

Various concentrations of the anti-B7-1 antibody (in duplicate) werepreincubated with 1×10⁴ CHO/hB7-1 cells for 1 hour at 37° C. in 100 μLRPMI complete medium (RPMI 1640, 10% FBS, 100 U/mL penicillin, 100 μg/mLstreptomycin) in a microtiter plate (flat bottomed, 96 well, Costar,Cambridge Mass.)

Results:

FIG. 12 shows the results of the inhibition of human CD28⁺ T cellproliferation by the murine and humanized forms of the anti-B7-1 mAbs.Both monoclonal antibodies exhibit dose dependent inhibition of B7-1driven proliferation of human T cells with similar IC50 (Inhibitoryconcentration 50%, amount of antibody required to inhibit maximal T cellproliferation by 50%) values of 110 pM (humanized anti-B7-1) and 220 pM(murine anti-B7-1) indicating that both antibodies were similar and veryeffective in inhibiting the B7-1 T cell co-stimulatory signal. Thisdemonstrates that the high affinity anti-B7-1 mAbs can block B7-1functionally by inhibiting or preventing the activation and/orproliferation of human T cells. These mAbs are expected to exhibitsimilar capability in in vivo use to inhibit T cell responses.

Example 16 Inhibition of Mixed Lymphocyte Reactions by Anti-B7-1 andAnti-B7-2 mAbs

Mixed lymphocyte reactions (MLR): Human normal peripheral bloodlymphocytes (PBL) (responders) were cultured with irradiated (2,500 cGy)normal donor PBL (stimulators) in RPMI 1640 containing 5%heat-inactivated human AB serum at 37° C. in 5% CO₂ at a finalconcentration of 10⁶ cells/mL. Where indicated, murine anti-hB7-1 ormurine anti-hB7-2 antibodies were added alone (10 μg/mL), in combination(10 μg/mL each), and in comparison with CTLA4Ig (10 or 20 μg/mL). Cellswere cultured in triplicate in microtiter plates in a final volume of200 μL and proliferation was assessed by [³H]-thymidine incorporationfor the last 16 hours of culture. Secondary MLR was performed using thecells derived from the primary MLRs as responders. These cells werewashed, cultured overnight, and restimulated as above using the same ordifferent, third party stimulator PBLs. No inhibitors were added to thesecondary MLRs.

Results:

The determinations shown in FIG. 13 were made by performing primaryone-way MLRs in the absence or presence of B7 inhibitors (anti-B7,CTLA4Ig). The proliferation was measured after 3, 4, or 5 days ofculture.

In the primary MLR, the additional anti-B7-1 mAb alone had no inhibitoryeffect indicating a minor role for B7-1 alone in driving proliferationof responder T cells. Anti-B7-2 alone inhibited T cell proliferation onall days tested at a level comparable to human CTL4Ig (hCTL4Ig), arecombinant protein known to bind to both B7-1 and B7-2. The combinationof anti-B7-1 and anti-B7-2 was the most effective inhibitor of T cellproliferation that completely inhibited this response on all daystested. The superior ability of the combined anti-B7-1 and anti-B7-2 toinhibit T cell proliferation, as compared to hCTL4Ig, reflects thehigher affinity of the anti-B7 mAbs for B7-1 and B7-2 as compared tohCTL4Ig. The combined anti-B7-1 and anti-B7-2 mAbs were betterinhibitors of T cell proliferation than anti-B7-2 alone, demonstratingthe need to block both stimulatory receptors to completely inhibit Tcell responses. These results show that complete blockade of the B7-1and B7-2 costimulators more completely abrogates alloresponsiveness inthe MLR. Accordingly, these results indicate that methods of treatmentincluding both anti-B7-1 and anti-B7-2 antibodies will be even moreeffective than either of the antibodies alone, especially where bothco-stimulatory molecules are functional. While the responder/stimulatorpair, described herein, was not sensitive to inhibition by anti-B7-1alone, some responder/stimulator pairs do exhibit moderate (0-50%)anti-B7-1 sensitivity.

To determine whether treatment with anti-B7 mAbs in the primary MLR hadresulted in the development of T cell hyporesponsiveness or anergy, theresponder T cells from the primary MLRs were tested in secondary MLRswhere the stimulators were either from the same donor as the primary MLRor from a third party. FIG. 14 shows that the responder T cells obtainedfrom the primary MLR that was treated with anti-B7-1 alone show fullproliferative responses to both the original sensitizing cells and tothird party cells when tested in a secondary MLR with noimmunosuppression indicating that blocking the B7-1 receptor alone bytreatment with the anti-B7-1 mAb had no tolerizing effect on theseresponding T cells. This is in contrast to the lack of response to theprimary stimulators seen in the secondary MLR when the primary MLR wastreated with anti-B7-2 alone. The results in FIG. 15 show that theresponder T cells from the primary MLR treated with anti-B7-2 alonefailed to respond to the same stimulators as used in the primary MLR butretained normal proliferative response to third party, unrelatedstimulators indicating that these responder T cells were renderedtolerant to the original stimulator PBLs by treatment with anti-B7-2 andthat the tolerization was specific for the stimulator antigens presentin the primary MLR. With this responder/stimulator pair, treatment withanti-B7-2 alone resulted in tolerance to the stimulator cells; however,with other responder/stimulator pairs, the induction of tolerance maynot be complete.

FIG. 16 shows that the responder T cells from the primary MLR treatedwith anti-B7-1 and anti-B7-2 failed to respond to the same stimulatorsas used in the primary MLR, but retained normal proliferative responseto third party, unrelated stimulators. This indicates that theseresponder T cells were rendered tolerant to the original stimulator PBLsby treatment with the combined anti-B7-1 and anti-B7-2. The resultsobtained with this responder/stimulator pair are typical for otherresponder/stimulator pairs in that tolerance induction is the rule.

Example 17 Inhibition of Immune Responses in Non-Human Primates byAnti-B7 mAbs; Inhibition of Anti-Tetanus Responses

Method:

Twelve tetanus naïve, 4-6 kg male Cynomolgus macaques (Macacafasicularis) were divided into four experimental groups of three animalsper group:

-   -   Group A; received 2 immunizations with 10 Lf Units (Flocculation        Units) i.m. tetanus toxoid on day 0 and 42 (controls).    -   Group B; received 10 mg/kg of each humanized anti-B7-1 (1F1) and        anti-B7-2 (3D1) i.v., at least 90 minutes before 10 Lf units        i.m. tetanus toxoid on day 0; tetanus toxoid immunization only        (without mAb pretreatment) on day 42 (Costimulation blockade        with primary immunization).    -   Group C; received tetanus toxoid immunization only (without mAb        pretreatment) on day 0; 10 mg/kg of each humanized anti-B7-1 and        anti-B7-2 i.v., at least 90 minutes before 10 Lf units i.m.        tetanus toxoid on day 42 (Costimulation blockade with secondary        immunization).    -   Group D; received 10 mg/kg of each humanized anti-B7-1 (1F1) and        anti-B7-2 (3D1) i.v., at least 90 minutes before 10 Lf units        i.m. tetanus toxoid on day 0; received 10 mg/kg of each        humanized anti-B7-1 and anti-B7-2 i.v., at least 90 minutes        before 10 Lf units i.m. tetanus toxoid on day 42 (Costimulation        blockade with primary and secondary immunization).

Serum samples for anti-tetanus antibody testing were collected on days0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, and 84.

Anti-Tetanus Antibody ELISA:

96-well ELISA plates were coated with tetanus toxoid lot TP-1002 at 4μg/ml. A four-log titration of serum samples was performed starting at1:100. Ab binding to tetanus was detected with a combination ofmonoclonal anti-human IgG and polyclonal goat anti-rhesus IgMHRP-conjugated antibodies, and developed with TNB substrate.

Results:

FIG. 17 shows the anti-tetanus IgM+IgG responses in monkeys immunizedwith tetanus toxoid and treated with the combined anti-B7-1 andanti-B7-2 mAbs.

Fourteen days after primary immunization, monkeys receiving tetanustoxoid only (Group A) had developed serum anti-tetanus antibody titersof log 3 to 3.5 indicating a normal immune response to the tetanusantigen. An increase anti-tetanus response was seen after the secondtetanus immunization on day 42. Monkeys treated with the combination ofanti-B7-1 and anti-B7-2 before both the primary and secondaryimmunization (Group D) lacked detectable antibody titers until at leastday 84. Monkeys treated with anti-B7-1 and anti-B7-2 before the primaryimmunization only (Group B) maintained an undetectable anti-tetanusantibody titer until at least day 42, and a low (less than log 2.5)until at least day 84. Pretreatment with anti-B7-1 and anti-B7-2 beforesecondary tetanus immunization suppressed the secondary antibodyresponse slightly (Group C vs. Group A). Therefore, the administrationof anti-B7 antibodies concurrent with exposure to a new antigen (tetanusimmunization) can prevent the development of a new antibody response andcan lessen the strength of a secondary response to the same antigen.Since aspects of the rejection of transplanted organs and manyautoimmune diseases involve the generation of antibody responses,treatment with humanized anti-B7 mAbs is useful in preventing organrejection and in treating autoimmune diseases.

Example 18 Serum Half-Life of Anti-B7 Antibodies in Non-Human Primates

The murine-anti-hB7-1 and murine-anti-hB7-2 mAbs were tested innon-human primates for serum half-life and target cell saturation. ThreeCynomolgus monkeys were dosed with one dose each of a combination of theanti-hB7-1 and anti-hB7-2 mAbs at 2, 8, or 20 mg each mAb/kg bodyweight. The monkeys were analyzed for mAb binding to PBMC (ProliferativeBlood Mononuclear Cells), serum mAb concentration, and primateanti-mouse antibody (PAMA) response (Table 7). PBMC saturation wasdetermined by flow cytometry (FACS) where PBMCs isolated from the bloodof mAb dosed primates were stained with goat-anti-murine Ig-PE (% invivo) or the PBMC were first reacted with the anti-hB7-1 and anti-hB7-2mAbs followed by detection with the goat-anti-murine Ig-PE (% ex vivo).The level of PBMC saturation at the various time points was calculatedby (% in vivo/% ex vivo)×100. This study shows that PBMC saturation forthe anti-hB7-1 and anti-B7-2 mAbs falls below 80% between days 4 to 6(mAbs @ 2 mg/ks), days 6 to 8 (mAbs @ 8 mg/kg), and days 13 to 20 (mAbs@ 20 mg/kg) depending upon mAb dose. Although not measured directly,there was no apparent dramatic decrease in the numbers of circulatingB7⁺ cells.

Serum half-lives of the anti-hB7-1 and anti-hB7-2 mAbs were measuredwith a specific ELISA for each mAb using hB7-1Ig or hB7-2Ig as targetand goat-anti-murine Ig HRP/ABTS for detection. These assays weresensitive to 400 ng/mL and 200 ng/mL for anti-hB7-2 and anti-hB7-1,respectively. PAMA responses were measured using a commerciallyavailable kit. The serum concentrations of the two anti-hB7 mAbs and thePAMA responses are shown at the individual dosage levels for each mAb.Both mAbs exhibit similar serum half lines of ˜48 hours as determined atall three dosage levels. Increasing mAb dosage increased serum mAbconcentrations by a comparable factor at all dosages and times tested.When dosed at 20 mg/kg, circulating mAb levels of >30 μg/mL were foundfor each mAb at 6 days post dosing.

PAMA responses to the anti-hB7-1 and anti-hB7-2 mAbs were low and werefirst measurable beginning 10 days after serum mAb levels had fallenbelow 10 μg/mL.

The serum half-life of humanized anti-human B7-2 and B7-1 antibodieswere also determined in Cynomolgus monkeys (n=6) dosed once with 10mg/kg of humanized anti-B7-2 antibody. Serum concentration was monitoredby specific ELISA assay for each antibody using HRP-anti human IgG2 andABTS.

FIG. 18 shows the serum concentration of the humanized B7-2 andhumanized B7-1 mAbs in Cynomolgus monkeys through 42 days after dosing.

The humanized anti-human B7-2 and anti-human B7-1 mAbs exhibited anextended serum half-life in Cynomolgus monkeys, as compared to a valueof approximately 2 days for the murine anti-human B7-2 and anti-humanB7-1 mAbs when dosed at the same level, demonstrating that the humanizedanti-human B7 mAbs were retained in circulation much longer than themurine anti-B7 mAbs.

TABLE 7 Results from the Preclinical primate studies Dose @ 2 mg eachmAb/kg Dose @ 8 mg each mAb/kg Dose @ 20 mg each mAb/kg Time PBL PBL PBLHours Anti-hB7-2 PAMA Saturation Anti-hB7-2 PAMA Saturation Anti-hB7-2PAMA Saturation (Days) μg/mL ng/mL % μg/mL ng/mL % μg/mL ng/mL % 0 BQLNeg. 0 BQL Neg. 0 BQL Neg. 0 .167 61 NT 206 NT 580 NT .5 59 NT 100 229NT 25 570 NT 65 1 52 NT 227 NT 527 NT 3 52 NT 100 230 NT 100 548 NT 1005 50 NT 139 NT 464 NT 8 44 NT 169 NT 412 NT 24 (1 D) 26 NT 70 103 NT 100286 NT 80 48 (2 D) 15 NT 100 59 NT 100 196 NT 100 96 (4 D) 2.4 NT 75 18NT 100 83 NT 100 144 (6 D)  BQL NT 95 3.9 NT 100 32 NT 100 192 (8 D) BQL NT 65 BQL NT 100 13 NT 100 240 (10 D) BQL NT BQL NT 3.9 NT 312 (13D) BQL Neg. 5 BQL Neg. 55 BQL Neg. 80 480 (20 D) 2908 10 4080 10 517 20684 (27 D) 1260 1460 1094 816 (34 D) BQL = Below Quantifiable Limit; NT= Not Tested

Example 19 Inhibition of Specific T-Cell Responses to Superantigens(Toxic Shock Syndrome Toxin-1; TSST-1)

NODscid mice were populated with human lymphocytes by the administrationof 10⁸ human PBLs. After 28 days, the mice were treated with TSST-1 (10mg, I.P.) with or without the treatment with the combined antibodies tohuman B7-1 and B7-2 (500 mg, I.V.). After 14 additional days, thepresence of human lymphocytes, T-cells, and TSST-1 specific T-cells(Vβ2-TCR-cells) in the peritoneal cavity was measured by FACS usingantibodies specific for human CD45, CD4, and human Vβ2-TCR.

TABLE 8 Human Addition T-cells (%) TSST-1 Anti-B7-1 + Anti-B7-2 TotalVβ2⁺ − − 10.2 3.9 + − 27.4 12.0 + + 23.4 3.8Results:

Table 8 shows the proportion of total human T cells and V_(β)2⁺-TCRhuman T cells (TSST-1 specific) found in the peritoneal cavity ofhu-NODscid mice. Treatment with TSST-1 greatly increased the percentageof human T cells and of TSST-1 specific human T cells (V_(β)2⁺) in thehuNOD-scid mice. Treatment with the anti-human B7-1 and B7-2 mAbsmoderately diminished the total human T cell response and completelyinhibited the expansion of the TSST-1 specific human T cells indicatingthat the anti-B-7 mAbs could effectively inhibit human T cellsuperantigen mediated responses.

The teachings of all the references, patents and/or patent applicationscited herein are incorporated herein by reference in their entirety.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of inhibiting the interaction of a first cell bearing CD28and/or CTLA4 with a second cell bearing B7-1, comprising contacting saidsecond cell with an effective amount of a humanized immunoglobulinmolecule having binding specificity for B7-1, said immunoglobulincomprising a constant region of human origin and a variable region,wherein said variable region comprises: a) one or more complementaritydetermining regions derived from the 1F1 mouse monoclonal antibody (ATCCAccession No. PTA 263) and b) one or more framework regions of humanorigin.
 2. A method of inducing lack of responsiveness to an antigen inan individual having a transplanted organ, tissue, or cell comprisingadministering an effective amount of a humanized antibody having bindingspecificity for B7-1, said antibody comprising a constant region ofhuman origin and a variable region, wherein said variable regioncomprises: a) one or more complementarity determining regions derivedfrom the 1F1 mouse monoclonal antibody (ATCC Accession No. PTA 263) andb) one or more framework regions of human origin.
 3. A method forreducing transplant rejection in an individual having a transplantedorgan, tissue, or cell, comprising administering a therapeuticallyeffective amount of a humanized antibody having binding specificity forB7-1, said antibody comprising a constant region of human origin and avariable region, wherein said variable region comprises: a) one or morecomplementarity determining regions derived from the 1F1 mousemonoclonal antibody (ATCC Accession No. PTA 263) and b) one or moreframework regions of human origin.
 4. The method of claim 3, wherein thehumanized antibody is administered with a pharmaceutical carrier.
 5. Themethod of claim 2, wherein the immunoglobulin is administered in apharmaceutically acceptable carrier.
 6. A method of inhibitingproliferation of a T cell comprising contacted the T cell with ahumanized antibody having binding specificity for B7-1, said antibodycomprising a constant region of human origin and a variable region,wherein said variable region comprises: a) one or more complementaritydetermining regions derived from the 1F1 mouse monoclonal antibody (ATCCAccession No. PTA 263) and b) one or more framework regions of humanorigin.
 7. The method of claim 1, wherein said immunoglobulin comprisesa light chain encoded by the amino acid of SEQ ID NO: 28 and a heavychain encoded by the amino acid sequence of SEQ ID NO:
 26. 8. The methodof claim 1, wherein said immunoglobulin comprises a constant regioncomprising a human IgG4 isotype.
 9. The method of claim 1, wherein saidimmunoglobulin comprises a constant region comprising a human IgG2M3isotype.
 10. The method of claim 1, wherein said one or more frameworkregions comprise an amino acid sequence derived from a framework regionof the III2R (SEQ ID NO: 44) heavy chain.
 11. The method of claim 1,wherein said one or more framework regions comprise an amino acidsequence derived from a framework region of the III2R (SEQ ID NO: 42)light chain.
 12. The method of claim 2, wherein said immunoglobulincomprises a light chain encoded by the amino acid sequence of SEQ ID NO:28 and a heavy chain encoded by the amino acid sequence of SEQ ID NO:26.
 13. The method of claim 2, wherein said immunoglobulin furthercomprises a constant region comprising a human IgG4 isotype.
 14. Themethod of claim 2, wherein said immunoglobulin further comprises aconstant region comprising a human IgG2M3 isotype.
 15. The method ofclaim 2 wherein said one or more framework regions comprise an aminoacid sequence derived from a framework region of the III2R (SEQ ID NO:44) heavy chain.
 16. The method of claim 2, wherein said one or moreframework regions comprise an amino acid sequence derived from aframework region of the III2R (SEQ ID NO: 42) light chain.
 17. Themethod of claim 3, wherein said immunoglobulin comprises a light chainencoded by the amino acid of SEQ ID NO: 28 and a heavy chain encoded bythe amino acid sequence of SEQ ID NO:
 26. 18. The method of claim 3,wherein said immunoglobulin further comprises a constant regioncomprising a human IgG4 isotype.
 19. The method of claim 3, wherein saidimmunoglobulin further comprises a constant region comprising a humanIgG2M3 isotype.
 20. The method of claim 3, wherein said one or moreframework regions comprise an amino acid sequence derived from aframework region of the III2R (SEQ ID NO: 44) heavy chain.
 21. Themethod of claim 3, wherein said one or more framework regions comprisean amino acid sequence derived from a framework region of the III2R (SEQID NO: 42) light chain.
 22. The method of claim 6 wherein saidimmunoglobulin comprises a light chain encoded by the amino acidsequence of SEQ ID NO: 28 and a heavy chain encoded by the amino acidsequence of SEQ ID NO:
 26. 23. The method of claim 6 wherein saidimmunoglobulin further comprises a constant region comprising a humanIgG4 isotype.
 24. The method of claim 6, wherein said immunoglobulinfurther comprises a constant region comprising a human IgG2M3 isotype.25. The method of claim 6, wherein said one or more framework regionscomprise an amino acid sequence derived from a framework region of theIII2R (SEQ ID NO: 44) heavy chain.
 26. The method of claim 6, whereinsaid one or more framework regions comprise an amino acid sequencederived from a framework region of the III2R (SEQ ID NO: 42) lightchain.
 27. The method of claim 1, wherein the binding affinity is about10⁹ M⁻¹.
 28. The method of claim 2, wherein the binding affinity isabout 10⁹ M⁻¹.
 29. The method of claim 3, wherein the binding affinityis about 10⁹ M⁻¹.
 30. The method of claim 6, wherein the bindingaffinity is about 10⁹ M⁻¹.
 31. A method of inhibiting the interaction ofa first cell bearing CD28 and/or CTLA4 with a second cell bearing B7-1,comprising contacting said second cell with an effective amount of ahumanized immunoglobulin having binding specificity for B7-1, saidimmunoglobulin comprising: a) a light chain which comprises one or moreCDRs derived from the 1F1 mouse monoclonal antibody (ATCC Accession No.PTA 263) and a framework region of human origin; and b) a heavy chainwhich comprises one or more CDRs derived from the 1F1mouse monoclonalantibody (ATCC Accession No. PTA 263) and a framework region of humanorigin.
 32. The method of claim 31, wherein said light chain comprisesthree CDRs derived from the 1F1 mouse monoclonal antibody (ATCCAccession No. PTA 263 and a framework region of human origin and saidheavy chain comprises three CDRs derived from the 1F1 mouse monoclonalantibody (ATCC Accession No. PTA 263) and a framework region of humanorigin.
 33. The method of claim 31 wherein said immunoglobulin comprisesa light chain encoded by the amino acid sequence of SEQ ID NO: 28 and aheavy chain encoded by the amino acid sequence of SEQ ID NO:
 26. 34. Themethod of claim 31 wherein said immunoglobulin further comprises aconstant region comprising a human IgG4 isotype.
 35. The method of claim31 wherein said immunoglobulin further comprises a constant regioncomprising a human IgG2M3 isotype.
 36. The method of claim 31, whereinthe binding affinity is about 10⁹ M⁻¹.
 37. A method of inducing lack ofresponsiveness to an antigen in a patient having a transplanted organ,tissue, or cell, comprising administering an effective amount of ahumanized immunoglobulin having binding specificity for B7-1, saidimmunoglobulin comprising: a) a light chain which comprises one or moreCDRs derived from the 1F1 mouse monoclonal antibody (ATCC Accession No.PTA 263) and a framework region of human origin; and b) a heavy chainwhich comprises one or more CDRs derived from the 1F1mouse monoclonalantibody (ATCC Accession No. PTA 263) and a framework region of humanorigin.
 38. The method of claim 37, wherein said light chain comprisesthree CDRs derived from the 1F1 mouse monoclonal antibody (ATCCAccession No. PTA 263) and a framework region of human origin and saidheavy chain comprises three CDRs derived from the 1F1 mouse monoclonalantibody (ATCC Accession No. PTA 263) and a framework region of humanorigin.
 39. The method of claim 37, wherein said immunoglobulincomprises a light chain encoded by the amino acid sequence of SEQ ID NO:28 and a heavy chain encoded by the amino acid sequence of SEQ ID NO:26.
 40. The method of claim 37, wherein said immunoglobulin furthercomprises a constant region comprising a human IgG4 isotype.
 41. Themethod of claim 37, wherein said immunoglobulin further comprises aconstant region comprising a human IgG2M3 isotype.
 42. The method ofclaim 37, wherein the binding affinity is about 10⁹ M⁻¹.
 43. A method ofreducing transplantation rejection in a patient having a transplantedorgan, tissue, or cell, comprising administering an effective amount ofa humanized immunoglobulin having binding specificity for B7-1, saidimmunoglobulin comprising: a) a light chain which comprises one or moreCDRs derived from the 1F1 mouse monoclonal antibody (ATCC Accession No.PTA 263) and a framework region of human origin; and b) a heavy chainwhich comprises one or more CDRs derived from the 1F1 mouse monoclonalantibody (ATCC Accession No. PTA 263) and a framework region of humanorigin.
 44. The method of claim 43, wherein said light chain comprisesthree CDRs derived from the 1F1 mouse monoclonal antibody (ATCCAccession No. PTA 263 and a framework region of human origin and saidheavy chain comprises three CDRs derived from the 1F1 mouse monoclonalantibody (ATCC Accession No. PTA 263) and a framework region of humanorigin.
 45. The method of claim 43, wherein said immunoglobulincomprises a light chain encoded by the amino acid sequence of SEQ ID NO:28 and a heavy chain encoded by the amino acid sequence of SEQ ID NO:26.
 46. The method of claim 43, wherein said immunoglobulin furthercomprises a constant region comprising a human IgG4 isotype.
 47. Themethod of claim 43, wherein said immunoglobulin further comprises aconstant region comprising a human IgG2M3 isotype.
 48. The method ofclaim 43, wherein the binding affinity is about 10⁹ M⁻¹.
 49. The methodof claim 1, wherein the one or more framework regions of human originderived from the III2R (SEQ ID NOS: 41-44) variable region.
 50. Themethod of claim 2, wherein the one or more framework regions of humanorigin derived from the III2R (SEQ ID NOS: 41-44) variable region. 51.The method of claim 3, wherein the one or more framework regions ofhuman origin derived from the III2R (SEQ ID NOS: 41-44) variable region.52. The method of claim 24, wherein the one or more framework regions ofhuman origin derived from the III2R (SEQ ID NOS: 41-44) variable region.53. The method of claim 1, wherein said-variable region comprises atleast two CDRs of the variable region of the 1F1 monoclonal antibody(ATCC Accession No. PTA 263).
 54. The method of claim 1, wherein saidvariable region comprises at least three CDRs of the variable region ofthe 1F1 monoclonal antibody (ATCC Accession No. PTA 263).
 55. The methodof claim 2, wherein said variable region comprises at least two CDRs ofthe variable region of the 1F1 monoclonal antibody (ATCC Accession No.PTA 263).
 56. The method of claim 2, wherein said variable regioncomprises at least three CDRs of the variable region of the 1F1monoclonal antibody (ATCC Accession No. PTA 263).
 57. The method ofclaim 3, wherein said variable region comprises at least two CDRs of thevariable region of the 1F1 monoclonal antibody (ATCC Accession No. PTA263).
 58. The method of claim 3, wherein said variable region comprisesat least three CDRs of the variable region of the 1F1 monoclonalantibody (ATCC Accession No. PTA 263).
 59. A method of inhibiting theinteraction of a first cell bearing CD28 and/or CTLA4 with a second cellbearing B7-1, comprising contacting said second cell with an effectiveamount of a humanized immunoglobulin having binding specificity forB7-1, said immunoglobulin comprising: a) a variable region derived fromthe 1F1 mouse monoclonal antibody ATCC Accession No. PTA 263), and b)one or more framework regions of a human antibody.
 60. A method ofinducing lack of responsiveness to antigen in a patient having atransplanted organ, tissue, or cell, comprising administering aneffective amount of a humanized immunoglobulin having bindingspecificity for B7-1, said immunoglobulin comprising: a) a variableregion derived from the 1F1 mouse monoclonal antibody (ATCC AccessionNo. PTA 263), and b) one or more framework regions of a human antibodywherein the immunoglobulin is administered in a carrier.
 61. A method ofreducing transplant rejection in a patient having a transplanted organ,tissue, or cell, comprising administering a therapeutically effectiveamount of a humanized antibody having binding specificity for B7-1, saidimmunoglobulin comprising: a) a variable region derived from the 1F1mouse monoclonal antibody (ATCC Accession No. PTA 263); and b) one ormore framework regions of a human antibody.